ADP-ribosylation of cyclophilin A by Pseudomonas aeruginosa exoenzyme S.
The virulence of the opportunistic pathogen Pseudomonas aeruginosa (Pa) is in part mediated by the type III secretion (TTS) of bacterial proteins into eukaryotic hosts. Exoenzyme S (ExoS) is a bifunctional Pa TTS effector protein, with GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities. Known cellular substrates of TTS-translocated ExoS (TTS-ExoS) ADPRT activity include proteins in the Ras superfamily and ERM family proteins. This study describes the ADP-ribosylation of a non-G-protein substrate of TTS-ExoS, cyclophilin A (CpA), a peptidyl-prolyl isomerase (PPIase). Four novel 17 kDa proteins (pI 6.5-6.8) were recognized in a proteomic screen of lysates of human epithelial cells that had been exposed to ExoS-producing Pa, but not an isogenic non-ExoS producing strain. The proteins were identified as isoforms of CpA using MALDI-TOF mass spectrometry and confirmed by Western blotting. Mutagenesis analysis identified arginine 55 and 69 of CpA as sites of ExoS ADP-ribosylation. Examination of the effect of ExoS ADP-ribosylation on CpA function found a moderate (19%) decrease in prolyl isomerization of a Xaa-Pro containing peptides. In comparison, GST-CpA co-immunoprecipitation studies found ExoS ADP-ribosylation of CpA to efficiently inhibit CpA binding to calcineurin/PP2B phosphatase. Our results support that ExoS ADP-ribosylates and affects the function of the cytosolic protein, CpA, with the predominant functional effect relating to interference of CpA-cellular protein interactions.
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Augustine A
AA
DiNovo
Kevin L
KL
Schey
William S
WS
Vachon
Eileen M
EM
McGuffie
Joan C
JC
Olson
Timothy S
TS
Vincent
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000378:metabolism*; D045744:Cell Line, Tumor; D021984:Cyclophilin A / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001427:Bacterial Toxins; D010446:Peptide Fragments; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D021984:Cyclophilin A