Strategies to Endow Phage-encoded Enzymes against Gram-negative Bacteria

The emergence of antimicrobial resistance has posed a great threat to the global health. Infections caused by multidrug resistant gram-negative (G-ve) bacteria are more difficult to treat compared with the gram-positive (G+ve) ones. In this thesis, I engineered bacteriophage-encoded enzymes, endolysins and depolymerase, to explore their uses as antibacterial enzymes against multidrug-resistant (MDR) G-ve bacteria. ; Bacteriophage endolysins (lysins, or murein hydrolases) are enzymes that bacteriophages utilize to degrade the cell wall peptidoglycans (PG) and subsequently disintegrate bacterial cells from within. Due to their muralytic activity, lysins are considered potential candidates to battle against antibiotic resistance. However, most lysins in their native form lack the capability of trespassing the outer membrane (OM) of G-ve bacteria. To turn the bacteriophage enzymes into antibacterial weapons against G-ve bacteria, endowing these enzymes the capability of accessing the PG substrate underneath the OM is thereby a critical step. Here we show that engineering the C-terminus of a globular lysin can drastically increase its OM permeabilization and the antibacterial activity against G-ve bacteria. Using LysAB2 as a model lysin, appending a cecropin A (CeA) peptide sequence at its C-terminus increases the antibacterial activity against a MDR strain of Acinetobacter baumannii by up to 100,000 folds. The engineered LysAB2, called LysAB2-KWK here, shows remarkable activity against stationary phase bacteria, prominent capability to disrupt biofilm formation, a broader spectrum against a range of G-ve bacteria, a modest tolerance to serum, and a prolonged storage life. The enzyme LysAB2-KWK also rescues the larva of the greater wax moth Galleria mellonella from A. baumannii infection through systemic administration. The antibacterial activity has been dissected as the combined effect of the PG degrading and the OM permeabilizing activity. ; Bacteriophage-encoded depolymerases are responsible for degrading ...