title: Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions.
abstract: BACKGROUND
  Pseudomonas aeruginosa, an increasingly prevalent opportunistic pathogen, utilizes a type III secretion system for injection of toxins into host cells in order to initiate infection. A crucial component of this system is PcrV, which is essential for cytotoxicity and is found both within the bacterial cytoplasm and localized extracellularly, suggesting that it may play more than one role in Pseudomonas infectivity. LcrV, the homolog of PcrV in Yersinia, has been proposed to participate in effector secretion regulation by interacting with LcrG, which may act as a secretion blocker. Although PcrV also recognizes PcrG within the bacterial cytoplasm, the roles played by the two proteins in type III secretion in Pseudomonas may be different from the ones suggested for their Yersinia counterparts.
  
  
  RESULTS
  In this work, we demonstrate by native mass spectrometry that PcrV and PcrG expressed and purified from E. coli form a 1:1 complex in vitro. Circular dichroism results indicate that PcrG is highly unstable in the absence of PcrV; in contrast, both PcrV alone and the PcrV:PcrG complex have high structural integrity. Surface plasmon resonance measurements show that PcrV interacts with PcrG with nanomolar affinity (15.6 nM) and rapid kinetics, an observation which is valid both for the full-length form of PcrG (residues 1-98) as well as a form which lacks the C-terminal 24 residues, which are predicted to have low secondary structure content.
  
  
  CONCLUSIONS
  PcrV is a crucial component of the type III secretion system of Pseudomonas, but the way in which it participates in toxin secretion is not understood. Here we have characterized the interaction between PcrV and PcrG in vitro, and shown that PcrG is highly unstable. However, it associates readily with PcrV through a region located within its first 74 amino acids to form a high affinity complex. The fact that PcrV associates and dissociates quickly from an unstable molecule points to the transient nature of a PcrV:PcrG complex. These results are in agreement with analyses from pcrV deletion mutants which suggest that PcrV:PcrG may play a different role in effector secretion than the one described for the LcrV:LcrG complex in Yersinia.
authors.affiliation: Institut de Biologie Structurale (CNRS/CEA/UJF), 41 rue Jules Horowitz, 38027 Grenoble, France. mnanao@ibs.fr
authors.forename: Max
authors.initials: M
authors.lastname: Nanao
authors.forename: Sylvie
authors.initials: S
authors.lastname: Ricard-Blum
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Di Guilmi
authors.forename: David
authors.initials: D
authors.lastname: Lemaire
authors.forename: David
authors.initials: D
authors.lastname: Lascoux
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Chabert
authors.forename: Ina
authors.initials: I
authors.lastname: Attree
authors.forename: Andréa
authors.initials: A
authors.lastname: Dessen
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D052899:Pore Forming Cytotoxic Proteins; D011550:Pseudomonas aeruginosa / Q000472:pathogenicity*; D016415:Sequence Alignment
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D046911:Macromolecular Substances; D052899:Pore Forming Cytotoxic Proteins; C118546:antigen V, Pseudomonas