Investigating new systems for integrity of the bacterial outer membrane
Lead Research Organisation:
University of Oxford
Department Name: Sir William Dunn Sch of Pathology
Abstract
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.
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.
Technical Summary
Antimicrobial resistant bacteria pose a significant threat to human health. The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier against harmful molecules, such as antibiotics. Understanding the mechanisms behind OM biogenesis is essential to explore new targets for drug design. In order to build and maintain their OM, bacteria must transport hydrophobic molecules, including lipids and proteins, across the aqueous periplasm. However, many of these mechanisms are poorly understood.
My research will focus on the AsmA protein family, which are predicted to transport molecules across the periplasm to build or maintain the OM. AsmA proteins are conserved across bacteria, with homologues in humans and plants, indicating they play an important role that has been maintained throughout evolution. Furthermore, bacteria lacking AsmA proteins show severe loss of OM homeostasis. This can result in loss of virulence, decreased antibiotic resistance, and even loss of viability. However, the structures and functions of AsmA proteins remain remarkably understudied, highlighting an important area for further research.
I propose to explore the function of the six AsmA proteins in E. coli using a combination of structural biology, protein biochemistry and bacterial genetics. I will solve the structure of AsmA proteins using cryo-EM. Furthermore, I will determine which components of the OM AsmA proteins directly interact with, to infer what they traffic. This will be achieved using a combination of pull-downs, mass spectrometry, and site-specific crosslinking. Finally, I will study the function of AsmA proteins at the cellular level, by constructing combinatorial gene knockouts and exploring phenotypes of these strains (e.g. cell morphology changes).
Overall, this research will increase our understanding of fundamental bacterial membrane biology, while identifying potential new candidates for the treatment of antimicrobial resistant bacterial infections.
My research will focus on the AsmA protein family, which are predicted to transport molecules across the periplasm to build or maintain the OM. AsmA proteins are conserved across bacteria, with homologues in humans and plants, indicating they play an important role that has been maintained throughout evolution. Furthermore, bacteria lacking AsmA proteins show severe loss of OM homeostasis. This can result in loss of virulence, decreased antibiotic resistance, and even loss of viability. However, the structures and functions of AsmA proteins remain remarkably understudied, highlighting an important area for further research.
I propose to explore the function of the six AsmA proteins in E. coli using a combination of structural biology, protein biochemistry and bacterial genetics. I will solve the structure of AsmA proteins using cryo-EM. Furthermore, I will determine which components of the OM AsmA proteins directly interact with, to infer what they traffic. This will be achieved using a combination of pull-downs, mass spectrometry, and site-specific crosslinking. Finally, I will study the function of AsmA proteins at the cellular level, by constructing combinatorial gene knockouts and exploring phenotypes of these strains (e.g. cell morphology changes).
Overall, this research will increase our understanding of fundamental bacterial membrane biology, while identifying potential new candidates for the treatment of antimicrobial resistant bacterial infections.
