Enzyme‐driven bacillus spore coat degradation leading to spore killing

Abstract

The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. 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 germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable.

Germination of Spores of Bacillus Species: What We Know and Do Not Know

“…While the inhibition or stimulation of spore germination in applied settings is somewhat off the focus of this review, these topics have attracted renewed interest in recent years because of the need for effective decontamination regimens for spores of organisms such as C. difficile and B. anthracis but without the concerns unique to the food industry. Thus, there are some recent reports of promising results in using a germination step prior to spore decontamination for promoting inactivation of B. anthracis and C. difficile spores (80)(81)(82), as well as enzymatic spore coat removal by lytic enzymes such as lysozyme to allow spore killing (83). Several compounds have also been identified that may be effective in inhibiting the germination or outgrowth of spores of organisms such as B. anthracis and C. difficile (84)(85)(86)(87)(88), and perhaps compounds analogous to these could be useful in applied settings.…”

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan

“…Endotoxin measurements were carried out using Pyrotell gel‐clot assay (Associates of Cape Cod, USA) as per manufacturer's instructions. Trinitrobenzene sulfonic acid (TNBS) assay was used to determine free amine content of GAG solution using L‐alanine as standard, as described previously …”

“…Trinitrobenzene sulfonic acid (TNBS) assay was used to determine free amine content of GAG solution using Lalanine as standard, as described previously. 34…”

Binding domains of Bacillus anthracis phage endolysins recognize cell culture age‐related features on the bacterial surface

“…For example, endolysins are expressed during the lytic phage cycle to destroy the host bacterial cell wall and release the viral progeny . This process involves targeting covalent bonds within the peptidoglycan layer of the bacterial cell wall . After a critical number of target sites have been hydrolyzed, the target bacterium disintegrates …”

“…7 This process involves targeting covalent bonds within the peptidoglycan layer of the bacterial cell wall. 8,9 After a critical number of target sites have been hydrolyzed, the target bacterium disintegrates. 10,11 Despite the fact that bacteria rapidly mutate to develop resistance against conventional antibiotics, 12,13 they are Elena E. Paskaleva and Ruchir V. Mundra contributed equally to the work.…”