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Research at Mass General
A recent research study by the research team in the lab of Marcia B. Goldberg, MD, sought to identify the human proteins required for bacterial pathogens to infect human cells.
Link to study
(Responses provided by Marcia Goldberg, MD, and Brian Russo, PhD)
To cause disease, most bacterial pathogens interact with human cells. These interactions require both bacterial and human factors. Human factors required for bacterial virulence are poorly characterized. The goal of our project was to identify human proteins required for bacterial pathogens to infect human cells.
Our work focuses on understanding how Shigella flexneri, a major cause of diarrhea, causes disease. To identify human genes that are required for Shigella infection, we tested the contribution to infection of nearly every human gene using a comprehensive approach known as a genetic screen. We identified that Shigella flexneri requires a structural protein in human cells known as an intermediate filament. Using a variety of cell biology and molecular biology approaches, we showed that intermediate filaments are required for initial steps in bacterial attachment to cells.
To identify human genes required for Shigella infection, we took a comprehensive approach that allowed us to test the contribution to infection of nearly every human gene. Our approach was unique because it built on the discovery of human-derived cells that contain only one copy of genetic material, which at the time, was the only way to efficiently generate a pool of human-derived cells that contained mutations in nearly every human gene.
To understand the stage of infection at which Shigella required intermediate filaments, we used novel cell biology-based assays to monitor the activity of pores formed by the bacteria in cell membranes and to assess docking of the bacteria onto the cells, as occurs in human infection. These assays were used to show that intermediate filaments were necessary for Shigella to dock onto cells.
To establish infection, many bacterial pathogens deliver bacterial proteins (effector proteins) into human cells. Effector proteins alter a variety of processes inside human cells; these alterations are responsible for many of the manifestations of the infection. Thus, effector proteins and their proper delivery are critical to the process of infection in human tissue. Initially, pathogens sit outside the cell, but they must deliver their proteins into the inside of the cell; to achieve this, they use a specialized apparatus known as a type 3 secretion system. More than 30 bacterial pathogens use this system. From outside the cell, pathogens using this system first make a pore in the cell membrane; they then deliver effector proteins through the pore into the inside of the cell. Our data define critical steps of this process at a molecular level. Our data show that after pore formation, one of the pore proteins (IpaC) interacts with intermediate filaments. The interaction with intermediate filaments allows the bacteria to dock to the pore and deliver its effectors through the pore and into the inside of the human cell. Although our work focused predominantly on one bacterial pathogen, Shigella, we also present data indicating that these events occur for many other bacterial pathogens.
A Mass General research team has discovered that interaction between structural proteins within the host cell called intermediate filaments and IpaC, a bacterial protein that forms the pore through the host cell membrane, is required for T3SSs to dock onto the pores and secrete effector proteins into the host cell. (Goldberg Laboratory, MGH Division of Infectious Diseases)
Our results demonstrate that pore formation on its own is not sufficient for the delivery of bacterial effector proteins into cells and show that following pore formation, bacteria must dock to the pore in a process that requires intermediate filaments. We have therefore shown that docking is a separate step from effector protein delivery; this discovery will facilitate future investigations into how these processes occur.
Our current work describes that intermediate filaments are required for Shigella flexneri to dock to the pore formed by the bacterium in the human cell membrane. We know that intermediate filaments are required, but we do not know how the interaction between the intermediate filaments and the pore protein allows the bacteria to dock to the pore. Future work will investigate how the intermediate filaments support bacterial docking to the pore and how this triggers the delivery of bacterial effector proteins into the cell.
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