Engineering of Phage-Derived Lytic Enzymes: Improving Their Potential as Antimicrobials

Abstract

Lytic enzymes encoded by bacteriophages have been intensively explored as alternative agents for combating bacterial pathogens in different contexts. The antibacterial character of these enzymes (enzybiotics) results from their degrading activity towards peptidoglycan, an essential component of the bacterial cell wall. In fact, phage lytic products have the capacity to kill target bacteria when added exogenously in the form of recombinant proteins. However, there is also growing recognition that the natural bactericidal activity of these agents can, and sometimes needs to be, substantially improved through manipulation of their functional domains or by equipping them with new functions. In addition, often, native lytic proteins exhibit features that restrict their applicability as effective antibacterials, such as poor solubility or reduced stability. Here, I present an overview of the engineering approaches that can be followed not only to overcome these and other restrictions, but also to generate completely new antibacterial agents with significantly enhanced characteristics. As conventional antibiotics are running short, the remarkable progress in this field opens up the possibility of tailoring efficient enzybiotics to tackle the most menacing bacterial infections.

Keywords: antibacterial; antibiotic resistance; antimicrobial; antimicrobial resistance; bacteriophage; endolysin; lysin; lytic enzyme; peptidoglycan hydrolase.

Figure 1

Natural context of action of phage lytic enzymes (PLEs). (a) Virion-associated lysins (VALs) promote a local digestion of the cell wall (CW) peptidoglycan to assist penetration of the phage tail tube and passage of the viral DNA to the host cell cytoplasm. After phage genome expression, infected cells must lyse to release the newly-formed virus particles. This is achieved thanks to the peptidoglycan-degrading activity of endolysins; (b) Most known endolysins gain access to the CW compartment through the holin channels (c-endolysins); (c) Some, however, are exported (e-endolysins) via host cell machineries (e.g., the bacterial Sec system). Holin-mediated dissipation of the cytoplasmic membrane proton-motive force (pmf) is an essential requirement for activation of e-endolysins, while it may also potentiate the lytic activity of c-endolysins (see text).

Figure 2

Basic structure of the bacterial cell wall peptidoglycan. The possible enzymatic activities of PLEs and the bonds they cleave are indicated. Typically, PLEs carry one or two catalytic domains displaying one of the indicated enzymatic activities. m-DAP is found in the peptide chains of the peptidoglycan of most Gram-negative bacteria, Bacillus spp. and Listeria spp., which present also direct m-DAP-D-Ala bonding between adjacent stem peptides. In most Gram-positive bacteria, m-DAP is replaced by L-Lys. Cross-linking between this residue and D-Ala of a neighbor peptide chain usually occurs by an interpeptide bridge of variable amino acidic composition (X). Despite some variation observed among isolates of the same bacterial species, examples of X bridges are (Gly)₅ found in Staphylococcus aureus, L-Ala-L-Ala in Enterococcus faecalis and Streptococcus pyogenes, D-Asp in E. faecium, and L-Ser-L-Ala in S. pneumoniae. The D-Ala residue in light blue may be lost after peptidoglycan maturation.

Figure 3

Domain architecture of endolysins (a) and VALs (b) that have been explored as enzybiotics, from Gram-positive and Gram-negative systems. CD, catalytic domain; CWBD, cell wall binding domain. The cardinals indicate the copy number of CW binding motifs composing the CWBD. The “n” letter indicates that a variable number of CW binding motifs may compose the CWBD (2 to 7 copies). These may be present either as tandem repetitions (in monomeric enzymes) or as oligomers when the CWBD subunit is independently produced by in-frame, alternative start sites (see text). The subunits of hetero-oligomeric endolysins are separated by the “+” sign. The presented VALs are from phages infecting Pseudomonas aeruginosa [56,57], S. aureus [58,59,60,61], and E. faecalis [62]. * Ply187 was firstly described as an endolysin. Schemes of phage lytic enzymes are not drawn to scale.