A C-terminal domain targets the Pseudomonas aeruginosa cytotoxin ExoU to the plasma membrane of host cells.
ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.
Rabin
Shira D P
SD
Department of Microbiology/Immunology, Northwestern University, 303 East Chicago Ave., Chicago, IL 60611, USA.
Veesenmeyer
Jeffrey L
JL
Bieging
Kathryn T
KT
Hauser
Alan R
AR
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D002462:Cell Membrane / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism; D021381:Protein Transport; D014162:Transfection
D001426:Bacterial Proteins; D010446:Peptide Fragments; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D010741:Phospholipases A