Enzyme-driven approaches to decontaminate bacillus cells and spores

Abstract

Bacillus cells have an exposed germ cell wall and are vulnerable to irreversible enzymatic hydrolysis. However, under environmental stress, they form recalcitrant spores. Spores are highly resistant to a range of chemical and physical assaults and to the best of our knowledge, a biocatalytic sporicidal system has never been developed before. To that end, we have developed and characterized an environmentally benign biocatalytic treatment against bacillus spores. A multilayered, cross-linked coat envelops the germ cell wall and confers the spores with resistance to lytic enzymes. Taking cues from nature, we identified proteases which degrade the formidable coat layer. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating spore germination. The germinated spores were further shown to be vulnerable to bacteriolytic enzymes resulting in effective spore killing.

Bacillus species are the causative agent of a wide spectrum of diseases ranging from food-borne illnesses to inhalation anthrax. Cell lytic enzymes (active component of bacteriophages) have recently attracted attention as a therapeutic alternative to antibiotics. We have focused on lytic enzymes with bactericidal activity against B. anthracis and B. cereus. Despite their exquisite selectivity and high potency, the inability to effect complete cell killing has restricted enzyme application in real-world settings. We attempted to fill this gap by fundamentally understanding the inherent limitation in enzyme-driven cell killing. Moreover, bactericidal activity of lytic enzymes such as PlyPH is strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary, non-dividing cells, thus limiting its application. We employed an in silico approach to identify diverse phage lysins with different binding targets within the bacterial cell wall. Specifically we identified Plyβ, an enzyme with improved cell binding capability and broad-based bactericidal activity.

To sum up, besides developing and characterizing enzyme based sterilization treatments against both cells and spores, we have addressed issues that potentially limit bacterial killing. These insights can be translated towards rationally engineering enzyme-based decontaminants to effect greater killing and subsequently be employed for environmental decontamination.