Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid

Maria B Nowakowska,Brigitte G Dorner,Maren Krüger,Rongsheng Jin,Adina Przykopanski,Katja Selby,Miia Lindström,Andreas Rummel,Nadja Krez,Martin B Dorner,Nigel P Minton

doi:10.1038/s41598-022-05008-1

Abstract

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

Highlights

The family of Botulinum neurotoxins (BoNTs) molecules, belonging to the zinc metalloprotease family, is divided into nine serotypes from A to G, an F/A hybrid serotype called H, and X, with a number of sub-serotype variants called subtypes[7,8,9,10,11]
A full-length, catalytically inactive BoNT/Ai carrying the mutations E224Q within the HExxH motif and R363A and Y366F within the RxxY motif displayed no detectable neurotoxicity in the mouse phrenic nerve hemidiaphragm (MPN) assay at 30,000-fold higher concentrations than wild-type BoNT/A27
Utilizing the CRISPR/Cas9 tool[41], we introduced point mutations into the genome of the Beluga Ei strain, altering the amino acid residues of the zinc-binding motif (638A > C resulting in E213A within HExxH + E motif) and the transition state stabilization motif (1042A > G, 1043G > C, 1044G > A, 1052A > T resulting in R348A and Y351F within RxxY motif)

Summary

Introduction

The family of BoNT molecules, belonging to the zinc metalloprotease family, is divided into nine serotypes from A to G, an F/A hybrid serotype called H, and X, with a number of sub-serotype variants called subtypes[7,8,9,10,11]. BoNT/Ai was used to resolve the crystal structure of the medium-sized (290 kDa) complex comprising BoNT/Ai and its non-toxic non-hemagglutinin N TNH27 These studies provide a solid base for constructing inactive BoNT mutants of other serotypes, as their catalytic motifs are strictly c onserved[18,28]. There is lack of evidence of sufficient genetic or physiological relationship between the non-toxigenic Clostridium spp. and C. botulinum Group II strains, limiting the reliability of Clostridium spp. as surrogates Another approach in circumventing the toxicity problem was bont/e gene deletion utilizing an allelic exchange approach[37]. Toxin production and other physiological processes in C. botulinum appear linked[38,39], which render bont knockout strains unreliable physiological surrogates

Methods

Results

Conclusion