IN-SITU CHARACTERIZATION OF CLOSTRIDIUM-BOTULINUM NEUROTOXIN SYNTHESIS AND EXPORT

A monoclonal antitoxin/colloidal gold probe and sequential centrifugat ion were used to study synthesis, translocation and export of Clostrid ium botulinum strain 62A neurotoxin (NT). Exponential growth occurred after 5 h of anaerobic incubation of spores and continued for 15-16 h. NT was detected at 15 h using the probe and transmission electron mic roscopy (TEM), 2 h earlier than the first detection by the mouse bioas say. During exponential growth, the probe localized NT primarily in th e cytoplasm, on the inner side of the cytoplasmic membrane and in the cell wall. During stationary and death phases, the NT was located with in the cytoplasm, cell wall and extracellularly. NT was released from the cell during cell wall exfoliation. Cells retained NT after repeate d gelatin-phosphate washes and sequential centrifugations, consistent with the TEM observation that the NT is bound to the cell wall. These observations indicate that the process of CI. botulinum type A NT prod uction follows a sequence of synthesis, translocation across the cytop lasmic membrane and export through the cell wall.