Cryo-scanning electron microscopy of Bordetella pertussis adenylate cyclase toxin embedded in lipid vesicles

L. Bumba¹, J. Nebesarova², P. Sebo¹

¹Institute of Microbiology,Videnska 1083, 142 20 Praha 4, Czech Republic

² Biological Centre, Czech Academy of Sciences, Branišovská 31, 370 05 Ceske Budejovice, Czech Republic

Email: bumba#biomed.cas.cz

The adenylate cyclase toxin (CyaA, ACT or Hly-AC) of Bordetella pertussis is a protein with a molecular mass of 200 kDa that belongs to RTX (repeat in toxin) protein family of bacterial pore-forming toxins. The CyaA toxin spontaneously inserts into eukaryotic membranes and it translocates its catalytic N-terminal domain into the cytosol, where catalyzes unregulated conversion of cellular ATP to cAMP. The C-terminal RTX hemolytic moiety accounts for binding of CyaA to target cells, for translocation of the adenylate cyclase domain and for making the cell membrane permeable by cation-selective pores. There are indirect evidences that CyaA could act either as a monomer or dimer/oligomer. In order to determine functional unit of CyaA in lipid bilayer we used high resolution cryo-scanning electron microscopy to visualize CyaA on the surface of lipid vesicles. CyaA was incubated with the suspension of liposomes and washed in bicarbonate buffer to remove non-specifically bound proteins. The CyaA- bound liposomes were quickly frozen in liquid nitrogen and left to evaporate in a Gatan Alto 2500 cryo-holder connected to a Jeol JSM-7401F field emission scanning electron microscope. CyaA molecules were seen as single protrusions with homogenous distribution on the surface of liposomes. The protrusions had an approximate diameter of about 15 nm indicating CyaA to be in monomeric form.