Recombinant design of the enzymatically active domain of phage Enc34 endolysin to improve its activity against Gram-negative bacteria.

Abstract

Endolysins are bacteriophage-encoded peptidoglycan-degrading enzymes with potential applications for treating multidrug-resistant bacterial infections. While exogenously applied endolysins are active against Gram-positive bacteria in their native form, Gram-negative bacteria are protected from such activity of most native endolysins by an outer membrane. However, it was shown that recombinant endolysins can be designed to efficiently lyse Gram-negative bacteria from without as well. During our previous efforts, we purified and structurally characterized the enzymatically active domain (EAD) of phage Enc34 endolysin. In this work, we investigated the lytic potential of products resulting from different variants of fusions involving this EAD with a panel of selected antimicrobial peptides. A set of constructs was generated and expressed in Escherichia coli cells. While most such recombinant proteins accumulated intracellularly, some of them could lyse cells from within and appear in the expression medium. The fusion protein variants produced were purified and tested for their bactericidal activity against Gram-negative bacteria. The best candidate caused rapid degradation of E. coli XL1-Blue cells during the first minutes after addition, reducing the viable cell count more than three-fold. We believe that these results might be helpful in the design of new antibacterial tools.