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 would like to determine in molecular detail how some of these pathogens enter human cells. Second, we want to analyze factors the bacteria encode that allow them to survive and grow within the ordinarily hostile environment of human cells. Finally, we are analyzing how these microorganisms circumvent the host immune system. Our main approach is to develop genetic and biochemical techniques to study the behavior of these microorganisms after contact with host cells in culture. This approach provides insights into basic processes that are applicable to numerous pathogenic microorganisms.
To investigate the molecular mechanism of bacterial binding and entry into host cells, as well as the host cell defense against these processes, we are analyzing the bacterium Yersinia pseudotuberculosis, an organism that causes an intestinal disease that often precedes infection of multiple organ systems. 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.
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. We have used several large-scale discovery strategies to determine that at least 120 different bacterial proteins are transported into the host cell via this bridge, and many of these proteins contribute to formation of the replication compartment.
One of the transported proteins, LidA, is a large protein that forms a coiled-coil structure and appears to bind membrane vesicles that originate from the host endoplasmic reticulum (ER). It does this by associating with members of the Rab family of proteins that are found in the host macrophage. Members of this family, when activated, control the transport, docking, and fusion of membrane vesicles to target compartments. A second transported bacterial protein, SidM, activates a Rab protein. When the activation activity of SidM is combined with the binding activity of LidA, the two bacterial proteins are able to tether macrophage vesicles to beads. The tethering event mimics processes observed during growth of the bacterium in macrophages. A third protein, SdhA, is necessary to prevent the killing of host cells byL. pneumophila. In the absence of SdhA, L. pneumophila cannot grow in macrophages because the macrophages die.
Yersinia pseudotuberculosis Regulation of Mammalian CellsY. 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. We have been interested in determining how the microorganism induces entry and manipulates signaling processes in host cells. Entry of Yersinia into host cells is promoted by invasin, a 108-kDa protein on the surface of the bacterium that binds host receptor molecules. The cell-binding region of invasin consists of two tightly associated domains sitting atop three other domains, arrayed like eggs balancing on top of each other. This five-domain array of eggs is presented to the mammalian cell for initial binding of the bacterium.
The cell-binding region of invasin recognizes at least five different receptors. Called integrins, these proteins had been previously identified by investigators interested in a variety of mammalian cell-adhesion processes, such as immobilizing cells to the matrix that supports them. Several mammalian signaling proteins in addition to integrins are required for bacterial uptake. Much of our recent work has been devoted to understanding how the bacterium manipulates these signaling proteins, because bacterial alteration of these proteins is critical for allowing uptake into cells during the initial disease process and for ensuring that the bacterium is outside cells at later times during disease. One such signaling protein is Rac1, which controls the cytoskeleton, a cell structure that is necessary for bacterial uptake. We used fluorescence resonance energy transfer (FRET) to show that shortly after the bacteria bind the target cell, the Rac1 protein is recruited to the site of bacterial binding and undergoes a conformational shape change that activates the protein and is necessary for bacterial uptake. As the infection proceeds, there appears to be a complex interplay between invasin and other Yersinia proteins, called Yops, that results in either inactivation of Rac1 or a change in the intracellular locale of Rac1. Our current work is devoted to determining how these potentially antagonistic proteins collaborate to promote disease.
We have also focused on determining how Y. pseudotuberculosis can initiate growth in the intestine of an animal and eventually find its way into deep organ sites. We have shown that to cause systemic disease, bacteria must first grow in the intestine, where they acquire enhanced virulence potential. Formerly, researchers had thought that disease-causing bacteria in the intestine first enter intestinal cells and then grow in nearby lymph nodes prior to moving to different sites in the animal via the bloodstream. By using bacterial and mouse mutants that block the ability of Y. pseudotuberculosis to enter intestinal cells, we found that the bacteria can find a different way to move to deep organs, by directly penetrating through the intestine and bypassing entry into the neighboring lymph nodes. Once across the intestine, the bacterium associates with immune cells. We are determining how the bacterium controls these cells and interferes with the host immune system.
A grant from the National Institute of Allergy and Infectious Diseases provided support for the work on Yersinia.
As of January 03, 2007