Periodic Reporting for period 3 - SecNine (Mechanism of Type 9 Secretion: an unusual protein transport system involved in pathogenesis and motility)
Período documentado: 2022-09-01 hasta 2024-02-29
The bacterial cell envelope is the interface between a bacterium and the surrounding world. It acts as a barrier to separate the cell contents from the surroundings. At the same time, bacteria must be able to export proteins across this barrier in order to influence their environment. For example a disease-causing bacterium needs to secrete proteins that will affect the host organism. Protein export across the cell envelope is carried out by transporter systems which must move only the desired proteins but not other molecules.
One recently discovered protein transporter is called the Type IX Secretion System, or T9SS for short. It is found in a large group of bacteria called the Bacteroidetes. The T9SS is essential for some of these bacteria to cause disease, for example the organisms that cause severe dental disease (periodontitis) in humans and the organisms that cause important diseases of farmed fish. Other members of the Bacteroidetes are beneficial members of the bacterial community that colonises the roots of crop plants. In these bacteria the T9SS is required to build an apparatus that allows the bacteria to move by gliding across solid surfaces.
The T9SS is extremely complex and involves at least 20 different proteins. Some of the proteins are thought to build the transporter itself, whilst other proteins are involved in recognising substrate proteins, processing substrate proteins, and providing mechanical energy to power the transport process. The roles of other proteins are unclear. This project aims to determine the structure and mechanism of the T9SS components through a wide variety of cutting edge approaches. This information will be important in informing understanding of the role played by the T9SS in causing disease, in designing potential therapies that block operation of the T9SS, and in possible attempts to use the T9SS for industrial protein production.
In the main part of the project we aim to put tags on different proteins from the T9SS and pull on the tags to find out what other proteins they associate with. This will identify and isolate the complexes these proteins form in the T9SS. We then aim to take pictures of the complexes with an electron microscope and use this to work out the detailed three dimensional structure of the complexes. This will give us information about how the different complexes of the T9SS work. We will label the different complexes with different coloured fluorescent markers and use these to see which complexes assemble together in cells and whether their assembly changes during operation of the T9SS mechanism. Finally, we will label a transported protein with a fluorescent marker and attempt to watch it being transported out of the cell. This will provide detailed information about the mechanism of transport.
One recently discovered protein transporter is called the Type IX Secretion System, or T9SS for short. It is found in a large group of bacteria called the Bacteroidetes. The T9SS is essential for some of these bacteria to cause disease, for example the organisms that cause severe dental disease (periodontitis) in humans and the organisms that cause important diseases of farmed fish. Other members of the Bacteroidetes are beneficial members of the bacterial community that colonises the roots of crop plants. In these bacteria the T9SS is required to build an apparatus that allows the bacteria to move by gliding across solid surfaces.
The T9SS is extremely complex and involves at least 20 different proteins. Some of the proteins are thought to build the transporter itself, whilst other proteins are involved in recognising substrate proteins, processing substrate proteins, and providing mechanical energy to power the transport process. The roles of other proteins are unclear. This project aims to determine the structure and mechanism of the T9SS components through a wide variety of cutting edge approaches. This information will be important in informing understanding of the role played by the T9SS in causing disease, in designing potential therapies that block operation of the T9SS, and in possible attempts to use the T9SS for industrial protein production.
In the main part of the project we aim to put tags on different proteins from the T9SS and pull on the tags to find out what other proteins they associate with. This will identify and isolate the complexes these proteins form in the T9SS. We then aim to take pictures of the complexes with an electron microscope and use this to work out the detailed three dimensional structure of the complexes. This will give us information about how the different complexes of the T9SS work. We will label the different complexes with different coloured fluorescent markers and use these to see which complexes assemble together in cells and whether their assembly changes during operation of the T9SS mechanism. Finally, we will label a transported protein with a fluorescent marker and attempt to watch it being transported out of the cell. This will provide detailed information about the mechanism of transport.
We have trapped and characterised a transporter complex that provides information on how the transporter protein works.
We have determined structures of the motor that drives T9SS transport. These structures suggest that the motor operates by a mechanism in which ion flow causes one component of the motor to rotate inside the other component.
We have identified new proteins that are involved in modifying substrate proteins following transport through the T9SS.
We have determined structures of the motor that drives T9SS transport. These structures suggest that the motor operates by a mechanism in which ion flow causes one component of the motor to rotate inside the other component.
We have identified new proteins that are involved in modifying substrate proteins following transport through the T9SS.
We have developed a new method to monitor protein export from cells by the T9SS as it occurs and have used this method to show that the transport through the transporter takes less than a second.