Site-specific chemoenzymatic labeling of aerolysin enables the identification of new aerolysin receptors

PLoS One. 2014 Oct 2;9(10):e109883. doi: 10.1371/journal.pone.0109883. eCollection 2014.

Abstract

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.

Publication types

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

MeSH terms

  • Aeromonas hydrophila / chemistry
  • Aeromonas hydrophila / metabolism*
  • Amino Acid Sequence
  • Animals
  • Bacterial Toxins / analysis
  • Bacterial Toxins / metabolism*
  • Biotin / chemistry
  • CD59 Antigens / analysis
  • CD59 Antigens / metabolism*
  • Cell Line
  • Fluorescent Dyes / chemistry
  • Glypicans / analysis
  • Glypicans / metabolism*
  • Humans
  • Molecular Sequence Data
  • Optical Imaging
  • Pore Forming Cytotoxic Proteins / analysis
  • Pore Forming Cytotoxic Proteins / metabolism*
  • Receptors, Urokinase Plasminogen Activator / analysis
  • Receptors, Urokinase Plasminogen Activator / metabolism*

Substances

  • Bacterial Toxins
  • CD59 Antigens
  • Fluorescent Dyes
  • Glypicans
  • Pore Forming Cytotoxic Proteins
  • Receptors, Urokinase Plasminogen Activator
  • aerolysin
  • Biotin