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Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract.

The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of Bacillus anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report for the first time a detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B. anthracis when added exogenously. CwlJ1 was active on both decoated spores and spore cortical fragments. Through liquid chromatography-mass spectrometry analysis of the digested cortical fragments, we determined that CwlJ1 was a thermostable N-acetylmuramoyl-L-alanine amidase. CwlJ1 mainly recognized large segments of glycan chains in the cortex instead of the minimal structural unit tetrasaccharide, with specificity for muramic acid-δ-lactam-containing glycan chains and preference for the tetrapeptide side chain. Unlike most amidases, CwlJ1 did not appear to contain a divalent cation, as it retained its activity in the presence of EDTA. This study shines some light on the mechanism of spore germination, and provides increased insight into the development of sporicidal enzyme systems for decontamination of B. anthracis and other related bacteria., (© 2015 Wiley Periodicals, Inc.)

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.