Novel Lytic Enzyme of Prophage Origin from <i>Clostridium botulinum</i> E3 Strain Alaska E43 with Bactericidal Activity against Clostridial Cells

• Main Authors: Agnieszka Morzywolek, Magdalena Plotka, Anna-Karina Kaczorowska, Monika Szadkowska, Lukasz P. Kozlowski, Dariusz Wyrzykowski, Joanna Makowska, Jerel J. Waters, Steven M. Swift, David M. Donovan, Tadeusz Kaczorowski
• Format: Article
• Language: English
• Published: MDPI AG 2021-09-01
• Series: International Journal of Molecular Sciences
• Subjects: <i>N</i>-acetylmuramoyl-<span style="font-variant: small-caps">l</span>-alanine amidase; <i>Clostridium botulinum</i>; endolysin; prophage; lipoteichoic acid
• Online Access: https://www.mdpi.com/1422-0067/22/17/9536
• ISSN: 1661-6596; 1422-0067

<i>Clostridium botulinum</i> is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus <i>Clostridium</i>. Bioinformatics analysis revealed in the genomes of several <i>Clostridium</i> species genes encoding putative <i>N</i>-acetylmuramoyl-<span style="font-variant: small-caps;">l</span>-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of <i>C. botulinum</i> E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from <i>E. coli</i> Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera <i>Clostridium</i>, <i>Bacillus, Staphylococcus,</i> and <i>Deinococcus</i>, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a <i>C. botulinum</i> Group II prophage, which can potentially be used to control this important pathogen.