title: A leucine-rich motif targets Pseudomonas aeruginosa ExoS within mammalian cells.
abstract: Type III cytotoxins contribute to the ability of bacterial pathogens to subvert the host innate immune system. ExoS (453 amino acids) is a bifunctional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96 to 232 comprise a Rho GTPase activating protein domain, while residues 233 to 453 comprise a 14-3-3-dependent ADP-ribosyltransferase domain. An N-terminal domain (termed the membrane localization domain [MLD]) targets ExoS to the Golgi-endoplasmic reticulum (Golgi-ER) of mammalian cells. This study identifies an amino acid motif that is responsible for the membrane binding properties of the MLD. Deletion mapping showed that the MLD included a symmetrical leucine-rich motif within residues 51 to 77 of ExoS. The terminal dileucines and internal leucines and an isoleucine within the MLD, but not charged or other hydrophobic residues, targeted a reporter protein to the Golgi-ER region of HeLa cells. Mutations of the leucines within the MLD did not affect type III secretion or translocation into HeLa cells but limited the ability of ExoS to ADP-ribosylate Ras GTPases. Mutations of charged residues within the MLD did not affect type III secretion, delivery into HeLa cells, or the ability of ExoS to ADP-ribosylate Ras GTPases. The organization of the leucines within the MLD of ExoS is different from that of previously described leucine-rich motifs but is present in several other bacterial proteins. This implies a role for intracellular targeting in the efficient targeting of mammalian cells by type III cytotoxins.
authors.affiliation: Medical College of Wisconsin, Microbiology and Molecular Genetics, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Barbieri
mesh_terms: D036002:ADP Ribose Transferases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D020727:ADP-Ribosylation Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001427:Bacterial Toxins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000382:microbiology; D003603:Cytotoxins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000378:metabolism / Q000472:pathogenicity*; D017384:Sequence Deletion; D018631:ras Proteins / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D003603:Cytotoxins; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D020727:ADP-Ribosylation Factors; D018631:ras Proteins; D007930:Leucine