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Molecular Genetics of Intracellular Microorganisms

Research Summary

Ralph Isberg is interested in understanding how bacterial pathogens enter and grow within host cells and how pathogens that initiate diseases in one locale can spread to other sites in the host.

Many pathogenic species of bacteria are capable of causing diseases by colonizing and growing within human hosts, using tactics that avoid normal immune responses. As part of a general strategy to establish an infectious niche, a variety of microorganisms cause diseases by entering and growing inside human cells soon after encounter. Bacteria that establish infections in this manner are called intracellular microorganisms. Among the diseases they cause are tuberculosis and the most common types of sexually transmitted and foodborne diseases found in the industrialized world.

Our objectives are to investigate three important aspects of the lifestyle of pathogenic microorganisms. First, we want to analyze factors the bacteria encode that allow them to survive and grow within the ordinarily hostile environment of human cells. Second, we are analyzing how pathogens circumvent the host immune system. Finally, we are interested in how pathogens set up a microbial community within tissues, with particular emphasis on understanding how a single pathogen can divide itself into several regulatory subpopulations. Our main approach is to develop genetic and biochemical techniques to study the behavior of these microorganisms after contact with host cells in culture and growth in mouse tissues. These approaches provide insights into basic processes that are applicable to numerous pathogenic microorganisms. 

To investigate intracellular growth, we are analyzing Legionella pneumophila, the causative agent of Legionnaire's disease pneumonia. The strategies used for intracellular growth of the bacterium are similar to those of a wide range of intracellular microorganisms. To investigate community behavior of bacteria within tissues we are analyzing Yersinia pseudotuberculosis, a relative of the plague bacterium that causes gastroenteric disease that proceed deep tissue invasion.

Legionella pneumophila Growth in Phagocytic Cells 
L. pneumophila 
causes a variety of diseases in humans, including Legionnaire's disease pneumonia. The bacterium grows in lung tissues after encounter with its human host. Its favorite habitat is within alveolar macrophages, cells that normally kill invading microorganisms. Macrophages kill or inhibit the growth of microorganisms by internalizing pathogens and sequestering them in vacuole compartments, which in turn fuse with lysosomes filled with antibacterial factors. L. pneumophila is able to prevent the introduction of the antibacterial lysosomal components into this site during the earliest times after the interaction of the bacterium with its host cell, and this allows the microorganism to grow in this compartment.

We are interested in determining how L. pneumophila is able to establish and grow within this protective niche, called a replication vacuole. To this end, we identified the Dot/Icm complex by isolating mutants that fail to form the replication compartment. The Dot/Icm complex acts as a bridge in the bacterial membrane for the purpose of transporting proteins into the host macrophage, and these transported bacterial proteins instruct the formation of the replication vacuole. At least 300 different bacterial proteins are transported into the host cell via this bridge, and many of these proteins contribute to formation of the replication compartment.

We found that although the Dot/Icm is required for growth in macrophages, almost all are dispensable for growth in these cells, with the exception of two highly conserved proteins, SdhA and MavN. SdhA, is necessary to maintain the integrity of the membrane compartment surrounding the bacterium. In the absence of this protein, the compartment releases bacteria into the macrophage cytosol, which degrades the bacteria, sending a danger signal the initiates macrophage death. MavN is an iron transporter that is placed directly in the vacuole membrane, and in its absence, the bacteria starve of iron. Our work has been focused on the detailed mechanisms of action of these two bacterial proteins.

Yersinia pseudotuberculosis Community Behavior During Growth in Tissues 
Y. pseudotuberculosis causes disease after contaminated food products are ingested. In animals, the bacterium first enters intestinal cells, but as the infection proceeds, it is found outside of cells growing in intestine-associated lymph nodes as well as in the liver and spleen. When the bacteria grow in the liver and spleen, they form microcolonies, surrounded by innate immune cells, such as neutrophils and macrophages.   Bacteria within these microcolonies separate themselves into distinct subpopulations, each having unique transcriptional programs based on their location relative to host cells, with bacteria in host cell contact responding to the neighboring host cell and a diffusible nitric oxide (NO) product (Figure 1). We are currently investigating the dynamics of the interactions that occur between these two subpopulations.

Grants from the National Institute of Allergy and Infectious Diseases provided support for the work on Yersinia and Legionella.

As of April 23, 2016

Scientist Profile

Investigator
Tufts University School of Medicine
Cell Biology, Microbiology