EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis

Abstract

Bacteriophage lytic enzymes, either endolysins or virion-associated lysins, have been receiving considerable attention as potential antibacterial agents, particularly for the combat of antibiotic-resistant Gram-positive pathogens. A conclusion that easily emerges from the careful analysis of a great number of reports on the field is that the activity of phage lytic enzymes is rarely studied in conditions that support robust growth of the target bacteria. Here, we report the construction and study of a chimerical lysin, EC300, which was designed to target and kill Enterococcus faecalis in conditions supporting vigorous bacterial growth. EC300 resulted from the fusion of a predicted M23 endopeptidase domain of a virion-associated lysin to the putative cell wall binding domain of a previously characterized amidase endolysin, both produced by the E. faecalis phage F170/08. This bacteriolysin-like protein exhibited a clear enhanced lytic activity over the parental endolysin when both were assayed in a rich bacterial growth medium. We demonstrate the killing efficacy of EC300 against growing cells of a panel of typed E. faecalis clinical strains with high level of antibiotic resistance. The possible reasons for the marked difference between the lytic performance of EC300 and that of the amidase are discussed.

More than a hole: the holin lethal function may be required to fully sensitize bacteria to the lytic action of canonical endolysins

“…Since the simple membrane de-energization by nutrient exhaustion or by permeation to small ions was sufficient to increase cendolysin activity, we concluded that it is the holin pmfdissipating action that boosts enzyme activity, and not its ability to promote the release of eventual cytoplasmic factors activating endolysins. It was previously shown for at least two other predicted c-endolysins that treatment of target cells with the pore-forming lantibiotic nisin resulted in a drastic enhancement of lytic activity (Garc ıa et al, 2010;Proença et al, 2015).…”

“…Most importantly, regardless the type of lysis strategy used by dsDNA phages, one rule seems to have been conserved: endolysins do not act (or act very poorly) until infected cells are first killed by the holin function. Having all this in mind, and considering our recent observations that bacteria actively growing in rich media are able to counteract the lytic action of externally added c‐endolysins (Proença et al ., , and unpublished results), we raised the possibility that holins may have an additional role in canonical lysis, that is, that of sensitizing bacteria to the lytic action of c‐endolysins. This putative function would be camouflaged by the essential role of holin holes, which provide the necessary conduit for c‐endolysin release to the CW.…”

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

“…Another chimeric protein composed of the endopeptidase domain of the VAL and the cell wall binding domain of the endolysin of Enterococcus faecalis phage F170/08 is the EC300 protein. The muralytic activity of EC300 showed enhanced activity over the original lysins against clinical, multidrug resistant E. faecalis strains (Proença et al 2015). …”

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile

“…The major classes of lysins attracting the most attention include endolysins, autolysins, virion-associated lysins (VALs), and class IIIa bacteriocins (or bacteriolysins). Both VALs and endolysins are encoded by bacteriophages and are essential for host infection and progeny release, respectively 1 2 3 4 5 . Autolysins are endogenous cell wall hydrolases and play active roles in cell wall synthesis and remodeling, and cell division 6 7 8 .…”