Dara W. Frank
Microbiology and Molecular Genetics
Medical College of Wisconsin
Director, Center for Infectious Disease Research
Research Focus: Genetic Regulation of Exotoxin Synthesis
PhD: University of Texas, Austin (1984)
Pseudomonas aeruginosa, which usually resides in soil and water environments, can infect compromised hosts to cause significant morbidity and mortality. The pathogenesis of this organism in humans involves multiple virulence factors whose expression patterns are integrated with growth and replication. The pathogenesis of P. aeruginosa is complex and involves the expression of cell surface molecules, sensing and signal transduction systems, motility, chemotaxis and the ability to secrete a number of destructive enzymes including proteases, lipases, neuraminidase, toxins and capsular material. My long-term objective is to understand the interplay between host and bacterial factors that lead to life-threatening infections with P. aeruginosa. We have focused on the relationship between expression of toxins injected by the type III system and Pseudomonas pathogenesis in acute infection models. The local as well as the systemic delivery of toxins emphasize the pathogenic potential of the bacterium. Our hypothesis is that delivery of the type III toxins inhibits the uptake and destruction of P. aeruginosa during the initial stages of colonization, allowing multiplication past the point of resolution by innate immune mechanisms. The outcome of infection is then determined by the extent of injury or compromised state of the host, the expression patterns of the various toxins, proteases and other destructive enzymes, and the host response.
To date four enzymes are injected into eukaryotic cells via the type III secretion/injection system, ExoS, ExoT, ExoY, and ExoU. ExoS and ExoT are bifunctional enzymes that possess GTPase activating protein (GAP) and ADP-ribosyltransferase activity. These enzymes are expressed together by a majority of the environmental and clinical isolates. GAP activity of ExoS/T inhibits phagocytosis and alters cytoskeletal structure in non-phagocytic cells. ExoS ADP-ribosyltransferase activity and type III-delivery is correlated with cytotoxicity. The target specificity of ExoS includes Ras, Rap, RalA, Rac, Rab, Cdc42 and ERM proteins (ezrin/radixin/moesin). It is postulated that cytotoxicity could be due to the inhibition of multiple signal transduction pathways and/or the disruption of actin cytoskeletal and receptor complexes. ExoY is an adenylyl cyclase that induces the accumulation of cytoplasmic pools of cAMP. Cytoplasmic cAMP is associated with the disruption of intercellular junctions in endothelial cells and the leakage of fluids, perhaps leading to increased inflammatory responses by the host. ExoU is a potent phospholipase. The injection of ExoU is acutely cytotoxic through the cleavage of acyl side chains from membrane-associated phospholipids. ExoS/T-encoded ADP-ribosyltransferase, ExoY-adenylyl cyclase and ExoU phospholipase are relatively inactive unless exposed to the intracellular environment. The combination of vectorial translocation and requirement for eukaryotic cofactors for enzymatic activity ensures that the bacterium delivers potent toxic proteins with little to no modification of its own substrates or targets. The current focus of my laboratory is to exploit the unique properties of the type III-delivered P. aeruginosa toxins to identify and characterize the eukaryotic cofactors for ExoU and ExoY and to understand the structure and function relationship between cofactor and enzyme activation. The model of structural and functional activities of ExoU is shown in Figure 1.
Room: TBRC C3960