Dara W. Frank
Professor
Microbiology and Molecular Genetics
Medical College of Wisconsin
Research Focus: Genetic Regulation of Exotoxin Synthesis
PhD: University of Texas, Austin (1984) |
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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.
Figure 1
Recent Publications
Maier TM, Casey MS, Becker RH, Dorsey CW, Glass EM, Maltsev N, Zahrt TC, Frank DW. Identification of Francisella tularensis Himar1-based Transposon Mutants Defective for Replication in Macrophages. Infect Immun. 2007 Aug 6; [Epub ahead of print]
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Frank DW, Zahrt TC. Genetics and genetic manipulation in francisella tularensis. Ann N Y Acad Sci. 2007 Jun;1105:67-97. Epub 2007 Mar 29.
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Lee VT, Pukatzki S, Sato H, Kikawada E, Kazimirova AA, Huang J, Li X, Arm JP, Frank DW, Lory S. Pseudolipasin A is a specific inhibitor for phospholipase A2 activity of Pseudomonas aeruginosa cytotoxin ExoU. Infect Immun. 2007 Mar;75(3):1089-98. Epub 2006 Dec 18.
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Sato H, Feix JB, Frank DW. Identification of superoxide dismutase as a cofactor for the pseudomonas type III toxin, ExoU. Biochemistry. 2006 Aug 29;45(34):10368-75.
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Pechous R, Celli J, Penoske R, Hayes SF, Frank DW, Zahrt TC. Construction and characterization of an attenuated purine auxotroph in a Francisella tularensis live vaccine strain. Infect Immun. 2006 Aug;74(8):4452-61.
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Kulasekara BR, Kulasekara HD, Wolfgang MC, Stevens L, Frank DW, Lory S. Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa. J Bacteriol. 2006 Jun;188(11):4037-50.
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Maier TM, Pechous R, Casey M, Zahrt TC, Frank DW. In vivo Himar1-based transposon mutagenesis of Francisella tularensis. Appl Environ Microbiol. 2006 Mar;72(3):1878-85.
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Sato H, Feix JB, Hillard CJ, Frank DW. Characterization of phospholipase activity of the Pseudomonas aeruginosa type III cytotoxin, ExoU. J Bacteriol. 2005 Feb;187(3):1192-5.
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Maier TM, Havig A, Casey M, Nano FE, Frank DW, Zahrt TC. Construction and characterization of a highly efficient francisella shuttle plasmid. Appl Environ Microbiol. 2004 Dec;70(12):7511-9.
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Sato H, Frank DW. ExoU is a potent intracellular phospholipase. Mol Microbiol. 2004 Sep;53(5):1279-90.
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Sayner SL, Frank DW, King J, Chen H, VandeWaa J, Stevens T. Paradoxical cAMP-induced lung endothelial hyperpermeability revealed by Pseudomonas aeruginosa ExoY. Circ Res. 2004 Jul 23;95(2):196-203. Epub 2004 Jun 10.
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Sato H, Frank DW, Hillard CJ, Feix JB, Pankhaniya RR, Moriyama K, Finck-Barbancon V, Buchaklian A, Lei M, Long RM, Wiener-Kronish J, Sawa T. The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU. EMBO J. 2003 Jun 16;22(12):2959-69.
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Miyata S, Casey M, Frank DW, Ausubel FM, Drenkard E. Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis. Infect Immun. 2003 May;71(5):2404-13.
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Frank DW, Vallis A, Wiener-Kronish JP, Roy-Burman A, Spack EG, Mullaney BP, Megdoud M, Marks JD, Fritz R, Sawa T. Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV. J Infect Dis. 2002 Jul 1;186(1):64-73. Epub 2002 Jun 14.
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Banwart B, Splaingard ML, Farrell PM, Rock MJ, Havens PL, Moss J, Ehrmantraut ME, Frank DW, Barbieri JT. Children with cystic fibrosis produce an immune response against exoenzyme S, a type III cytotoxin of Pseudomonas aeruginosa. J Infect Dis. 2002 Jan 15;185(2):269-70. No abstract available.
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Holder IA, Neely AN, Frank DW. PcrV immunization enhances survival of burned Pseudomonas aeruginosa-infected mice. Infect Immun. 2001 Sep;69(9):5908-10.
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Finck-Barbancon V, Frank DW. Multiple domains are required for the toxic activity of Pseudomonas aeruginosa ExoU. J Bacteriol. 2001 Jul;183(14):4330-44.
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Roy-Burman A, Savel RH, Racine S, Swanson BL, Revadigar NS, Fujimoto J, Sawa T, Frank DW, Wiener-Kronish JP. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J Infect Dis. 2001 Jun 15;183(12):1767-74. Epub 2001 May 17.
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Holder IA, Neely AN, Frank DW. Type III secretion/intoxication system important in virulence of Pseudomonas aeruginosa infections in burns. Burns. 2001 Mar;27(2):129-30.
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Moss J, Ehrmantraut ME, Banwart BD, Frank DW, Barbieri JT. Sera from adult patients with cystic fibrosis contain antibodies to Pseudomonas aeruginosa type III apparatus. Infect Immun. 2001 Feb;69(2):1185-8.
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Pederson KJ, Pal S, Vallis AJ, Frank DW, Barbieri JT. Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells. Mol Microbiol. 2000 Jul;37(2):287-99.
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Cowell BA, Chen DY, Frank DW, Vallis AJ, Fleiszig SM. ExoT of cytotoxic Pseudomonas aeruginosa prevents uptake by corneal epithelial cells. Infect Immun. 2000 Jan;68(1):403-6.
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Kurahashi K, Kajikawa O, Sawa T, Ohara M, Gropper MA, Frank DW, Martin TR, Wiener-Kronish JP. Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J Clin Invest. 1999 Sep;104(6):743-50.
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Coburn J, Frank DW. Macrophages and epithelial cells respond differently to the Pseudomonas aeruginosa type III secretion system. Infect Immun. 1999 Jun;67(6):3151-4.
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Olson JC, Fraylick JE, McGuffie EM, Dolan KM, Yahr TL, Frank DW, Vincent TS. Interruption of multiple cellular processes in HT-29 epithelial cells by Pseudomonas aeruginosa exoenzyme S. Infect Immun. 1999 Jun;67(6):2847-54.
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Pederson KJ, Vallis AJ, Aktories K, Frank DW, Barbieri JT. The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins. Mol Microbiol. 1999 Apr;32(2):393-401.
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Sawa T, Yahr TL, Ohara M, Kurahashi K, Gropper MA, Wiener-Kronish JP, Frank DW. Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat Med. 1999 Apr;5(4):392-8.
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Vallis AJ, Finck-Barbancon V, Yahr TL, Frank DW. Biological effects of Pseudomonas aeruginosa type III-secreted proteins on CHO cells. Infect Immun. 1999 Apr;67(4):2040-4.
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Vallis AJ, Yahr TL, Barbieri JT, Frank DW. Regulation of ExoS production and secretion by Pseudomonas aeruginosa in response to tissue culture conditions. Infect Immun. 1999 Feb;67(2):914-20.
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Contact Information
Email: frankd@mcw.edu
Phone: 414-955-8766
Room: TBRC C3960