title: The GAP portion of Pseudomonas aeruginosa type III secreted toxin ExoS upregulates total and surface levels of wild type CFTR.
abstract: BACKGROUND
  Pseudomonas aeruginosa (PA) infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST) ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP) and C-terminal ADP rybosyl transferase (ADPRT) activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP). In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP) mediates cellular toxicity.
  
  
  METHODS
  The effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o- Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system.
  
  
  RESULTS
  Transient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT) CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na(+)/K(+)ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K), known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of ∆F508CFTR.
  
  
  CONCLUSIONS
  The GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections.
authors: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 40207, USA.
authors: Deepali N
authors: DN
authors: Tukaye
authors: Sang-Ho
authors: SH
authors: Kwon
authors: Wiliam B
authors: WB
authors: Guggino
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004285:Dogs; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D061985:Madin Darby Canine Kidney Cells; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018942:Macrolides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C040929:bafilomycin A1; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D000254:Sodium-Potassium-Exchanging ATPase