Analysis of Bacterial Uptake & Growth within Mammalian Cells
The work in our laboratory is directed toward investigating the molecular mechanisms of uptake and intravacuolar growth in host cells by bacterial pathogens as well as understanding how the host innate immune response deals with pathogen attack. The investigation of the Yersinia pseudotuberculosis invasin protein has been the primary focus of our studies on uptake, and analyses of intravacuolar growth have been performed using Legionella pneumophila. We have also made recent inroads into understanding how the host responds to pathogen proteins. The most exciting recent developments are our identification of pathways in host cells targeted by L. pneumophila proteins and the characterization of a host immune response to Yersinia adhesive events.
Intracellular growth of Legionella pneumophila
L. pneumophila is a pathogen that has adapted to grow within amoebae, and also causes a severe pneumonia in humans known as Legionnaire’s disease. The bacterium avoids phagocyte killing by growing within a replication vacuole that initially bypasses the lysosomal network. The bacterium is internalized into a membrane-bound replication vacuole that recruits rough endoplasmic reticulum. In addition a number of host proteins that are involved in moving membrane from the endoplasmic reticulum to other compartments in the secretory apparatus are found associated with the replication vacuole. One of these host proteins is Rab1, a small GTPase involved in membrane docking and fusion.
Figure 1. This schematic shows the path of Legionella inside a cell as it replicates in membrane-bound compartment surrounded by rough endoplasmic reticulum. This is in contrast to a nonpathogen which is directed to the lysosome.
Formation of this replication vacuole requires the products of 26 dot/icm bacterial genes, which encode proteins that assemble into an apparatus that translocates proteins into target host cells. We have devoted considerable work to identifying proteins that are translocated via this apparatus, and there are now 193 of these proteins that have been identified. These translocated substrates appear to associate with cytoplasmic surface of the membranous compartment surrounding the bacteria. Our laboratory is attempting to construct a unified model for how these different bacterial proteins interact with host signaling pathways. Two of these proteins are SidM and LidA, both of which interact with Rab1 and are found nearby the replication compartment.
Figure 2. These immunofluorescence pictures show Legionella (in red) within a replication vacuole that associates with host Rab1 (left panel, green) or with the bacterial proteins SidM (green) and LidA (green).
Yersinia pseudotuberculosis control of host cell responses
Yersinia pseudotuberculosis is internalized by M cells overlying the intestinal Peyer's patches Shortly after this event, bacteria are found exclusively extracellularly due to the production of Yops, translocated bacterial proteins that antagonize phagocytosis. Our work demonstrated that the primary bacterial-encoded factor that allows uptake both into M cells and into cultured cells is the outer membrane protein invasin, which binds multiple β1 integrin receptors. Our recent work has demonstrated that a key signaling molecule downstream from the integrin is the small GTPase, Rac1, which directs actin rearrangements in response to bacterial binding. Much of the focus of our research is directed toward analyzing how Rac1 is manipulated by Yersinia.
Targeting of Rac1 by Y. pseudotuberculosis takes place at several levels. Invasin activates Rac1, putting it in the GTP bound state, while several Yop proteins inactivate Rac1 and prevent it from being involved in phagocytosing bacteria or directing a host innate immune response to the microorganism. One of the main tools we use to study Rac1 activation is Fluorescence Resonance Energy Transfer (FRET), which allows us to determine if different pools of Rac1 in the cell are activated or inactivated after Yersinia attack.
Figure 3. These pictures show our readout for Rac1 activation and images of Yersinia after it binds a host cell.
In the absence of the Yops, invasin activates Rac1 and generates a positive FRET signal (green) in the area near where the bacteria bindIn addition to our studies on our studies on manipulation of Rac1, we have been investigating the host immune response to Yersinia virulence factors. We have developed a model that the host cell recognizes common patterns that are presented to the immune system by pathogens, but which are not presented by nonpathogens. These patterns include the presence of specialized secretion systems as well as proteins translocated into host cells by the pathogen. We are currently pursuing this model by analyzing the interplay between Yops and invasin.