Bacteriophage endolysins as novel antimicrobials

Abstract

Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology.

Figure 1. Schematic representation of selected bacteriophage endolysins, illustrating their modular architecture

The scale bar indicates the number of amino acids. CBD: Cell wall binding domain; ChBD: Choline binding domain; Cpl-7: Cpl-7-like cell wall binding domain; SH3b: Bacterial Src homology 3 domain.

Figure 2. Structures of bacterial peptidoglycan types and cut sites of different peptidoglycan hydrolases

(A) A1γ type featuring a direct cross-link between m-DAP and d-Ala, as present in Listeria, Bacillus and most Gram-negative species. (B) Staphylococcus aureus peptidoglycan of the A3α type featuring a pentaglycine cross-bridge. (C) Streptococcal peptidoglycan (A3α) with an l-Ala–l-Ala interpeptide bridge. Sugar units are depicted as hexagons and amino acids as ovals. The different classes of peptidoglycan hydrolases are represented by different shapes and letters and their cut sites are indicated by arrows. Specific examples including the endolysins shown in Figure 1 and the bacteriocin lysostaphin are given next to the respective symbols. All five classes of enzymes are present in (A), whereas only selected examples are shown in (B & C). Triangles with E numbers in them represent different types of endopeptidases. A: N-acetylmuramoyl-l-alanine amidase; E1: l-alanoyl-d-glutamate endopeptidase; E2: d-glutamyl-m-DAP endopeptidase; E3: Interpeptide bridge-specific endopeptidase; E4: Glycylglycine endopeptidase; E5: d-alanoyl-glycine endopeptidase; E6: d-glutamyl-l-lysine endopeptidase; E7: d?-alanoyl-l-alanine endopeptidase; G: N-acetyl-β-d-glucosaminidase; L: Lytic transglycosylase; M: N-acetyl-β-d-muramidase.

Figure 3. Assays used to determine enzymatic and antimicrobial activity of endolysins

(A) Turbidity reduction assay. The change in optical density of a bacterial suspension upon addition of buffer as control (squares) or a defined amount of endolysin specific for the target cells (diamonds) is shown. The dashed line indicates the steepest slope of the resulting lysis curve [Schmelcher M, Unpublished Data]. (B) SDS-PAGE and zymogram. An SDS-PAGE assay of a protein preparation containing the target endolysin at an estimated purity >90% (prominent band) and multiple contaminant proteins (faint bands) is shown on the left, and a zymogram of the same preparation with substrate cells embedded in the gel is shown on the right. Lytic activity of the endolysin is indicated by a single cleared band [Roach D, Unpublished Data]. (C) Determination of the MIC. A twofold dilution series of an endolysin was mixed with target bacteria in growth medium and incubated overnight (bottom row). Clear wells (dark) indicate growth inhibition, with the last clear well representing the enzyme's MIC (boxed). Control rows containing no bacteria (top) or buffer without endolysin mixed with bacteria (middle) are also shown [Schmelcher M, Unpublished Data]. (D) Spot-on-lawn assay. Tenfold serial dilutions of a peptidoglycan hydrolase were spotted on a freshly plated lawn of exponentially growing target bacteria (10 μl per spot). After overnight incubation, cleared spots indicate lytic activity [Schmelcher M, Unpublished Data]. E: Endolysin preparation; M: Prestained molecular weight marker; OD_{600 nm}: Optical density measured at a wavelength of 600 nm.

Figure 4. Use of cell wall binding domains for specific differentiation and immobilization of target cells

(A) Differentiation of Listeria cells of two different strains by fluorescent microscopy using cell wall binding domains of different serovar specificities fused to either GFP or dsRed as fluorescent markers [Schmelcher M, Unpublished Data]. (B) Phase-contrast image of a Listeria cell immobilized on the surface of a cell wall binding domain-coated paramagnetic bead [Eichenseher F, Unpublished Data].