Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.
Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.
Lin
Guangyun
G
Department of Bacteriology, Food Research Institute, University of Wisconsin-Madison, Madison, WI 53706, USA.
Tepp
William H
WH
Pier
Christina L
CL
Jacobson
Mark J
MJ
Johnson
Eric A
EA
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D003014:Clostridium botulinum / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D005810:Multigene Family; D011817:Rabbits; D013194:Staining and Labeling / Q000379:methods
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D019274:Botulinum Toxins, Type A