Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947

Botulinum toxin is produced by Clostridium botulinum as a large toxin complex (L-TC) non-covalently assembled with a neurotoxin (NT), a non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33, and HA-17). In this study, the gene expressions of five individual L-TC components were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in C. botulinum serotype D strain 4947 (D-4947) during cell growth. Transcripts for the five component genes were successfully detected in the mid-exponential growth phase (6.5 h), reaching a maximum at the early stationary growth phase (12 h). The ratio of the mRNA transcripts of nt and ntnha was approximately 1:1, suggesting that nt and ntnha are bicistronically transcribed. On the other hand, the transcript levels of the ha genes were several-fold higher than those of nt and ntnha, although the mRNA transcript level of ha-33 was less than the other two ha subcomponent genes. The results based on qRT-PCR indicate that a shortage of HA-33 among the proteins associated with botulinum TC could explain the production by D-4947 of other smaller-sized L-TCs (610, 540 and 410 kDa) with fewer HA-33 molecules than the mature 650 kDa L-TC. Western blot analysis demonstrated that TC species in cell lysate were initially observed in the mid-exponential phase, while extracellular TCs were detected subsequently in the early stationary phase.