title: Pseudomonas aeruginosa ExoT Induces Mitochondrial Apoptosis in Target Host Cells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain Activity.
abstract: Pseudomonas aeruginosa is the most common cause of hospital-acquired pneumonia and a killer of immunocompromised patients. We and others have demonstrated that the type III secretion system (T3SS) effector protein ExoT plays a pivotal role in facilitating P. aeruginosa pathogenesis. ExoT possesses an N-terminal GTPase-activating protein (GAP) domain and a C-terminal ADP-ribosyltransferase (ADPRT) domain. Because it targets multiple non-overlapping cellular targets, ExoT performs several distinct virulence functions for P. aeruginosa, including induction of apoptosis in a variety of target host cells. Both the ADPRT and the GAP domain activities contribute to ExoT-induced apoptosis. The ADPRT domain of ExoT induces atypical anoikis by transforming an innocuous cellular protein, Crk, into a cytotoxin, which interferes with integrin survival signaling. However, the mechanism underlying the GAP-induced apoptosis remains unknown. In this study, we demonstrate that the GAP domain activity is both necessary and sufficient to induce mitochondrial (intrinsic) apoptosis. We show that intoxication with GAP domain results in: (i) JNK1/2 activation; (ii) substantial increases in the mitochondrial levels of activated pro-apoptotic proteins Bax and Bid, and to a lesser extent Bim; (iii) loss of mitochondrial membrane potential and cytochrome c release; and (iv) activation of initiator caspase-9 and executioner caspase-3. Further, GAP-induced apoptosis is partially mediated by JNK1/2, but it is completely dependent on caspase-9 activity. Together, the ADPRT and the GAP domains make ExoT into a highly versatile and potent cytotoxin, capable of inducing multiple forms of apoptosis in target host cells.
authors.affiliation: From the Department of Immunology/Microbiology.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Wood
authors.affiliation: From the Department of Immunology/Microbiology.
authors.forename: Josef W
authors.initials: JW
authors.lastname: Goldufsky
authors.affiliation: From the Department of Immunology/Microbiology.
authors.forename: Daniella
authors.initials: D
authors.lastname: Bello
authors.affiliation: From the Department of Immunology/Microbiology.
authors.forename: Sara
authors.initials: S
authors.lastname: Masood
authors.affiliation: From the Department of Immunology/Microbiology, Department of Internal Medicine, and Cancer Center, Rush University Medical Center, Chicago, Illinois 60612 Sasha_Shafikhani@rush.edu.
authors.forename: Sasha H
authors.initials: SH
authors.lastname: Shafikhani
mesh_terms: D036002:ADP Ribose Transferases; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D051023:BH3 Interacting Domain Death Agonist Protein; D000072224:Bcl-2-Like Protein 11; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D048055:Mitogen-Activated Protein Kinase 8 / Q000235:genetics / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D011552:Pseudomonas Infections / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C000606775:BCL2L11 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D000072224:Bcl-2-Like Protein 11; C477521:ExoT protein, Pseudomonas aeruginosa; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D051028:bcl-2-Associated X Protein; D036002:ADP Ribose Transferases; D048056:Mitogen-Activated Protein Kinase 9; D048055:Mitogen-Activated Protein Kinase 8; C506433:CASP9 protein, human; D053453:Caspase 9
keywords: BCL-2; Bax; Bid; Bim; ExoT; JNK; Pseudomonas aeruginosa (P. aeruginosa); apoptosis; bacterial toxin; c-Jun; c-Jun N-terminal kinase (JNK); c-Jun transcription factor; cytochrome c