Functional characterization of botulinum neurotoxin serotype H as a hybrid of known serotypes F and A (BoNT F/A).
A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5, while the C-terminus of the heavy chain (HC) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity toward SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.
†Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy NE, Atlanta, Georgia 30341, United States.
Suzanne R
SR
Kalb
†Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy NE, Atlanta, Georgia 30341, United States.
Jakub
J
Baudys
‡Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Enteric Diseases Laboratory Branch, 1600 Clifton Road, Atlanta, Georgia 30329, United States.
Brian H
BH
Raphael
‡Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Enteric Diseases Laboratory Branch, 1600 Clifton Road, Atlanta, Georgia 30329, United States.
Janet K
JK
Dykes
‡Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Enteric Diseases Laboratory Branch, 1600 Clifton Road, Atlanta, Georgia 30329, United States.
Carolina
C
Lúquez
‡Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases, Enteric Diseases Laboratory Branch, 1600 Clifton Road, Atlanta, Georgia 30329, United States.
Susan E
SE
Maslanka
†Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy NE, Atlanta, Georgia 30341, United States.
John R
JR
Barr
D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D003014:Clostridium botulinum / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry
D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096321:botulinum toxin type F