CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

Abstract

The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum

“…Group I strains were propagated in tryptone-peptone-glucose-yeast extract (TPGY) broth at 37 °C. Group II strains were grown in TPGY broth and a bi-phasic sporulation medium (Cooked Meat Medium Agar + TPGY) at 30 °C [ 26 ]. Finally, Group III strains were grown at 37 °C in TPGY supplemented with 0.2% ( w / v ) L-cysteine [ 51 ].…”

“…The sporulation strategies of C. botulinum are poorly described, but differences between spores of Group I and II strains are evident [ 26 ]. Dormancy and resistance to physical and chemical stressors allow spores to survive harsh environmental conditions, such as high temperature, aerobiosis, desiccation, and UV radiation [ 16 ], that would kill or damage vegetative cells.…”

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

“…Next, we verified the strains for BoNT production. We determined the concentrations of BoNT/Ei and BoNT/E in intracellular and extracellular culture fractions, respectively, using BoNT/E-directed sandwich ELISA based on the polyclonal capture antibody KE97 and the monoclonal BoNT/E-specific antibody E136 directed against the H N -domain of the toxin, unaltered in BoNT/Ei 43 . We additionally confirmed the ELISA-measured concentrations in densitometric analysis (Fig.…”

“…Culture aliquots were centrifuged (15 min; 6800 × g ; + 4 °C) and cell pellets re-suspended in PBS to extract the intracellular toxin. BoNT/E-directed sandwich ELISA was carried out as described 43 employing an affinity-purified polyclonal rabbit IgG KE97 as capture agent and the biotinylated mouse monoclonal antibody E136 directed against the H N -domain of BoNT/E as detector 58 . Purified BoNT/E (Metabiologics, Madison, WI, USA) was used as standard.…”

Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato

“…A possible role in virulence for type II-C CRISPR-Cas systems has been suggested ( Mir et al, 2018 ), but there is no experimental evidence for such function in Clostridium novyi sensu lato . Because C. botulinum genome engineering has been successful using CRISPR type II-B Cas9 ( Mertaoja et al, 2021 ), type II-C can also be explored as a native alternative for genome modification using a mini-plasmid strategy ( Pyne et al, 2016 ). This strategy may be advantageous, as modification of clostridia is cumbersome.…”