Bacteriophage endolysins and their use in biotechnological processes.

University of Ss. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Department of Biotechnology, J.Herdu 2, SK-917 01, Trnava, Slovakia. *Corresponding author: lenka.tisakova@savba.sk ABSTRACT Keywords: bacteriophage endolysins, EAD, CBD, applications INTRODUCTION Most of the tailed phages achieve correctly timed lysis by the consecutive use of two phage-encoded lysis proteins - endolysin and holin (Young et al., 2000; Loessner, 2005). All dsDNA phages produce a soluble, muralytic enzyme known as an endolysin. To degrade the cell wall, endolysins require a second lysis factor, a holin. Host cell lysis is mostly strictly regulated and exactly timed with the help of a holin. Holins are small hydrophobic proteins that are inserted into the cytoplasmatic membrane. At a genetically determined time in the terminal stage of the lytic cycle and upon a critical holin effector concentration and partial depolarization of the membrane, the holin monomers instantly assemble into oligomers and form membrane lesions “holes” through which the endolysins can then pass and lyse the bacterial cell wall (Vukov et al., 2003; Loessner, 2005; Fenton et al., 2010). Breach of the peptidoglycan (PG) layer results in osmotic lysis and cell death of the bacterium, thus enabling liberation of progeny virions (Schmelcher et al., 2012). Most known endolysins lack a secretory signal sequence and depend entirely on the cognate holin, which somehow permeabilizes the membrane, and is required for the endolysin to gain access to the murein for release to the PG (Loessner et al., 2002; Loessner, 2005). In most dsDNA phages the holin and endolysin genes cluster together, respectively, into the so called “lysis cassette” as part of the late transcribed genes (Fig. 1), although deviations from this spatial and temporal organization are found both in Gram-negative and Gram-positive systems. In addition to this basic lytic function, other phage-encoded proteins may work as auxiliary lysis factors (Young et al., 2000). In general, phage-encoded endolysins are easily identified by simple analysis of phage genomic sequences, due to the relatively high amino acid conservation observed within their catalytic domains (Sao-Jose et al., 2007). Bacteriophage endolysins are dsDNA bacteriophage-encoded peptidoglycan hydrolases (PGHs) which are synthesized in phage-infected bacterial cells during the late phase of gene expression at the end of multiplication cycle (Loessner, 2005). They are similar in structure and function to bacterial auto- and exo-lysins (Lopez et al., 1997; Shen et al., 2012) and also closely related to the similar family of mammalian PG recognition proteins (Low et al., 2011). Numerous studies have investigated the specificity of endolysins by assaying the cleavage sites on purified PG (Loessner et al., 1998; Navarre et al., 1999; Pritchard et al., 2004; Dhalluin et al., 2005; Fukushima et al., 2007; 2008; Mayer et al. 2008; 2011). Figure 1 Bacteriophage genome architecture. The diagram represents a typical template bacteriophage genome with genes involved in specific stages in phage development being grouped into modules (boxes). The specific genes/DNA sequences for holin and endolysin within the lysis module are indicated (bold text) (according to McGrath et al., 2004) ENDOLYSIN MODULAR ARCHITECTURE Generally, the structure of bacteriophage endolysins differ between those enzymes targeting Gram-positive and Gram-negative bacteria, reflecting the differences in the cell wall architecture between these major bacterial groups. Endolysins from a Gram-positive background have evolved to utilize a modular design. This is achieved by the combination of at least two distinct polypeptide modules (separated functional domains) that are dedicated two basic functions: substrate recognition and enzymatic hydrolysis, as depicted in Fig. 2. The N-terminal enzymatically active domain(s) = EAD generally harbor the catalytic activity (cleaving the specific bonds within the bacterial PG), whereas the C-terminal cell wall binding domain(s) = CBDs direct the enzymes to their substrates (Loessner et al., 2002; Fischetti, 2005; Loessner, 2005) and keep it tightly bound to cell wall debris after cell lysis, thereby likely preventing diffusion and subsequent destruction of surrounding intact cells that have not yet been infected by the phage (Loessner et al., 2002). Most of the endolysins studied to date are composed of at least two clearly separated functional domains (Loessner, 2005; Borysowski et al., 2006; Hermoso et al., 2007; Fenton et al., 2010; Fischetti, 2010; Schmelcher et al., 2012). However, endolysin structures are not necessary limited to only two modules (Diaz et al., 1990; Garcia et al., 1990), and the different architectures and domain orientations found in public databases are various (Nelson et al., 2012). Bacteriophage endolysins are peptidoglycan hydrolases, produced in the lytic system of bacteriophage in order to lyse host peptidoglycan from within and release virions into the environment. Phages infecting Gram-positive bacteria express endolysin genes with the characteristic modular structure, consisting of at least two functional domains: N-terminal enzymatically active domain (EAD) and C-terminal cell wall binding domain (CBD). CBDs specifically recognize ligands and bind to the bacterial cell wall, whereas EAD catalyze lysis of the peptidoglycan bonds. The reveal of endolysin modular structure leads to new opportunities for domain swapping, construction of chimeras and production of specifically engineered recombinant endolysins and their functional domains with the diverse biotechnological applications from without, such as in detection, elimination and biocontrol of pathogens, or as anti-bacterials in experimental therapy. ARTICLE INFO Received 21. 10. 2013 Revised 22. 11. 2013 Accepted 8. 1. 2014 Published 1. 2. 2014 Review