The presence of protective layers of spore cortex, spore coat and exosporium, endows bacterial spores with resistance against harsh chemical and biological treatments. A particularly relevant target of high economic, medical and biodefense importance is the spore-former Bacillus anthracis, the causative agent of anthrax. Current decontamination approaches involve the use of harsh chemicals such as bleach, vaporized hydrogen peroxide, ethylene oxide, which requires shutdown of contaminated facilities for months, further increasing the decontamination cost by several folds. As a result, there continues to be a need for the development of efficient and environmentally friendly treatments for deadly bacterial pathogens including B. anthracis. An attractive approach is to use highly selective bacteriolytic enzymes that target non-redundant features of bacteria making it extremely difficult for the pathogen to gain resistance. We have developed an approach to identify novel bacteriolytic enzymes by using amino acid sequence of a consensus binding domain as a probe toanalyze in silico the genome of B. anthracis Str. Ames. We characterized the mechanism of action of the the newly identifiede enzyme (AmiBA2446) on isolated cell wall peptidoglycan,by using LC-MS to analyze muropeptides released due to the enzymatic digestion of peptidoglycan. We evaluated the antimicrobial efficacy of enzyme against rapidly dividing cells as well germinating spores. We also demonstrated the selectivity of AmiBA2446 against B. anthracis in comparison to a panel of Bacillus species. We observed that the enzyme was exceptionally stable in aqueous solution for at least 4 months further expanding its potential for commercial application. We have also developed an �outside in� enzymatic decontamination strategy- from protease-mediated spore coat degradation, followed by spore cortex hydrolysis using cortex lytic enzymes, to germ cell wall lysis by bacteriolytic enzymes, thus demonstrating a mild and environmentally benign enzymatic approach for spore decontamination.

Enzyme-based targeted decontamination of <i>Bacillus</i> cells and spores

247th National Meeting (2014)

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Enzyme-based targeted decontamination of Bacillus cells and spores