Phage lysin that specifically eliminates Clostridium botulinum Group I cells

Zhen Zhang,Hannu Korkeala,Miia Lindström,Meeri Lahti,François P Douillard

doi:10.1038/s41598-020-78622-6

Abstract

Clostridium botulinum poses a serious threat to food safety and public health by producing potent neurotoxin during its vegetative growth and causing life-threatening neuroparalysis, botulism. While high temperature can be utilized to eliminate C. botulinum spores and the neurotoxin, non-thermal elimination of newly germinated C. botulinum cells before onset of toxin production could provide an alternative or additional factor controlling the risk of botulism in some applications. Here we introduce a putative phage lysin that specifically lyses vegetative C. botulinum Group I cells. This lysin, called CBO1751, efficiently kills cells of C. botulinum Group I strains at the concentration of 5 µM, but shows little or no lytic activity against C. botulinum Group II or III or other Firmicutes strains. CBO1751 is active at pH from 6.5 to 10.5. The lytic activity of CBO1751 is tolerant to NaCl (200 mM), but highly susceptible to divalent cations Ca2+ and Mg2+ (50 mM). CBO1751 readily and effectively eliminates C. botulinum during spore germination, an early stage preceding vegetative growth and neurotoxin production. This is the first report of an antimicrobial lysin against C. botulinum, presenting high potential for developing a novel antibotulinal agent for non-thermal applications in food and agricultural industries.

Highlights

Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin (BoNT)
CBO1751 was identified by prophage region analysis of the genome of C. botulinum Group I strain ATCC3502 using the PHASTER web server31. cbo1751, encoding a putative phage lysin, was found in one of two intact prophage regions
Bacteriophage lysins are the essential peptidoglycan-degrading proteins that are required by double-stranded DNA phages for the lysis of host bacterial cell wall and release of their viral progenies to extracellular space

Summary

Introduction

Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin (BoNT). Human botulism is predominantly associated with C. botulinum Group I and II. Clostridium perfringens is one of the most extensively studied anaerobic Gram-positive bacteria with characterized phage lysins and identified application potential. A series of further studies have significantly improved the application potential of these lysins in prevention of C. perfringens-associated food-borne illness and enteric diseases of domestic animals, or as a diagnostic tool in Scientific Reports | (2020) 10:21571. A phage lysin acting against C. botulinum would allow a novel non-thermal solution to control botulism hazards in foods or feeds. We report a putative phage lysin, CBO1751, identified in C. botulinum Group I strain ATCC3502. We demonstrate that recombinantly expressed CBO1751 has specific lytic activity against cells of C. botulinum Group I. We show that CBO1751 can effectively eliminate newly germinated cells, offering potential for early interruption of the pathogenic process of C. botulinum

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Conclusion