Detection of toxin translocation into the host cytosol by surface plasmon resonance.
AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1).  These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol.  Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4).  In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5).  The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process:  most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER(6-12).  To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)(13-15).  The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody.  The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin.  With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event.  The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor.  Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.
Department of Molecular Biology and Microbiology, University of Central Florida, USA.
Michael
M
Taylor
Tuhina
T
Banerjee
Neyda
N
VanBennekom
Ken
K
Teter
D002772:Cholera Toxin / Q000032:analysis / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020349:Surface Plasmon Resonance / Q000379:methods*; D014118:Toxins, Biological / Q000032:analysis / Q000378:metabolism*
D014118:Toxins, Biological; D002772:Cholera Toxin