Pseudomonas aeruginosa exoenzyme Y directly bundles actin filaments

J Biol Chem. 2020 Mar 13;295(11):3506-3517. doi: 10.1074/jbc.RA119.012320. Epub 2020 Feb 4.


Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin-bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY-F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.

Keywords: Pseudomonas aeruginosa; actin; actin bundling; bacterial pathogenesis; bacterial toxin; cytoskeleton; exoenzyme Y (ExoY); host-microbe interactions; host-pathogen interaction; protein-protein interaction; virulence factor.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Actin Cytoskeleton / metabolism*
  • Actin Cytoskeleton / ultrastructure
  • Actin Depolymerizing Factors / metabolism
  • Actins / chemistry
  • Actins / metabolism
  • Actins / ultrastructure
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Bacterial Proteins / ultrastructure
  • Glucosyltransferases / chemistry
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism*
  • Glucosyltransferases / ultrastructure
  • Membrane Glycoproteins / metabolism
  • Microfilament Proteins / metabolism
  • Models, Molecular
  • Mutation / genetics
  • Protein Binding
  • Protein Multimerization
  • Pseudomonas aeruginosa / enzymology*


  • Actin Depolymerizing Factors
  • Actins
  • Bacterial Proteins
  • Membrane Glycoproteins
  • Microfilament Proteins
  • plastin
  • ExoY protein, bacteria
  • Glucosyltransferases

Associated data

  • PDB/5XNW
  • PDB/3J0S
  • PDB/5YU8
  • PDB/3BYH