Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification

Strains ofClostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed thatC. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in theC. botulinum Hall A genome. DesignatedflaA1 andflaA2 , these open reading frames encode the major structural flagellins ofC. botulinum . Colony PCR and sequencing offlaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification offlaA1/A2 from type E strain Bennett identified a second flagellin gene,flaB . LC-MS analysis confirmed thatflaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by theflaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins ofC. botulinum to be diverse, the presence of theflaA1/A2 gene in all strains examined facilitates single locus sequence typing ofC. botulinum using the flagellin variable region.