Peptidoglycan-targeting lytic enzymes for antimicrobial applications

Abstract

Taking advantage of the superior properties of peptidoglycan-targeting lytic enzymes, we have explored their applications in three different systems: Gram-positive bacterial spores, Gram-positive bacterial cells, and Gram-negative bacterial cells. We have investigated the detailed mechanisms of the lytic enzymes and their interactions with the target peptidoglycan in these systems.

We have demonstrated the activities and properties of lytic enzymes in buffer conditions, in rich and poor media, and in intracellular environment. Covering both Gram-positive and Gram-negative bacteria with much focus on specific mechanisms, our work provides important guidance to the rational design of safe, stable, efficacious enzymatic systems for both environmental disinfection and medical treatment.

With Gram-negative bacteria, we have built programmed death into Escherichia coli for timed autolysis. By introducing a plasmid based lytic gene into E. coli, we have been able to lyse the cells from the inside when needed within 2 h. This approach has potential applications in gene or protein replacement therapy with live E. coli cells serving as delivery vehicles.

In the Gram-positive cell system, we have chosen Clostridium difficile as a model organism and have studied the mechanisms through which the C. difficile lytic enzyme CD11 loses activity in growth medium. We have found that the failure of CD11 to kill this bacterium in growth medium is not due to the inhibition of enzyme activity by the medium; rather, it is the lack of binding sites on the cell surface that renders the enzyme nonfunctional. Different medium compositions influence the cell surface properties greatly, and we have demonstrated that the cell surface hydrophobicity contributes to the cell-enzyme interactions. With promising therapeutic applications of bacterial lytic enzymes, our investigations benefit the proper design of highly efficient antibacterial enzyme systems for the disinfection in nutrient-rich regions like the human gut.

On the Gram-positive spore platform, we have shown that the cortex lytic enzyme CwlJ1 in Bacillus anthracis spores can trigger germination of decoated spores independently to release core materials, and the enzyme alone is sufficient to depolymerize the isolated cortical fragments, by presenting a thermostable, metal-independent N-acetlymuramoyl-L-alanine amidase activity. Despite the absence of a predicted binding domain, the enzyme has good specificity toward a particular structure of peptidoglycan, by targeting only large polysaccharide chains with muramic acid-δ-lactam residues while leaving the germ cell wall intact. These findings help to better understand the mechanism of spore germination, and will further facilitate the development of sporicidal enzymatic systems.