Bacteriophage endolysins — current state of research and applications

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Endolysins are phage-encoded enzymes that break down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle. Their action is tightly regulated by holins, by membrane arrest, and by conversion from their inactive to active state. Recent research has not only revealed the unexpected diversity of these highly specific hydrolases but has also yielded insights into their modular organization and their three-dimensional structures. Their N-terminal catalytic domains are able to target almost every possible bond in the peptidoglycan network, and their corresponding C-terminal cell wall binding domains target the enzymes to their substrate. Owing to their specificity and high activity, endolysins have been employed for various in vitro and in vivo aims, in food science, in microbial diagnostics, and for treatment of experimental infections. Clearly, phage endolysins represent great tools for use in molecular biology, biotechnology and in medicine, and we are just beginning to tap this potential.

Introduction

Endolysin is a rather generic term used to describe a range of bacteriophage-encoded peptidoglycan hydrolases, which are synthesized in phage-infected cells at the end of the multiplication cycle. These enzymes are also known as phage lysozymes, lysins, or muralytic/mureolytic enzymes. They are uniformly characterized by their ability to directly target bonds in the peptidoglycan (PG) layer of the bacterial cell wall; the result of this activity is degradation of the rigid murein layer and release of newly assembled virions by way of lysis.

An important issue is regulation of endolysin-mediated lysis of the infected host cells, and this has been intensively studied using phage λ and other Escherichia coli phages as prototypes (reviewed in [1]). Most of the tailed phages achieve correctly timed lysis by the consecutive use of two lysis proteins — endolysin and holin. Holins are small hydrophobic proteins that are encoded by the phage and insert into the cytoplasmic membrane (Figure 1). Within a genetically programmed time window, and upon a specific trigger event (critical holin effector concentration and partial depolarization of the membrane), the holin monomers instantly assemble into oligomers and form membrane lesions or holes through which the endolysins can then pass. The result is sudden cell lysis. Holins themselves can also be regulated by inhibitors, which interfere with intra-membrane assembly of the active holin. If present, the inhibitor is usually transcribed from the same reading frame as the holin, but features a functionally defective transmembrane domain. Besides transcription from the common dual AUG start motif, translation of the inhibitor can also be initiated at an intragenic start codon [2].

Most known endolysins lack a signal peptide sequence; they depend entirely on the cognate holins for release to the PG. However, recent studies have revealed the presence of a signal peptide in an Oenococcus oeni phage [3] and in the Lactobacillus plantarum phage Øg1e [4]. The endolysin of this phage requires the function of host sec for export. A particularly interesting case is the endolysin of E. coli phage P1, which features an N-terminal signal arrest release sequence [5, 6••]. Using this signal, the enzyme is exported to the membrane where it is arrested and processed by disulfide isomerization. Interestingly, the P1 holin was found to subsequently trigger and support release of the activated enzyme from the membrane [6••]. The lipid-containing virus PRD1 presents yet another case, in which the endolysin is associated with the viral membrane [7].

It is important to note that, with respect to Gram-positive cells, endolysins can also act as exolysins because the PG is, in most cases, accessible from without (Figure 2). This is not the case for Gram-negative cells, in which the presence of the outer membrane effectively prevents access by hydrophilic lytic enzymes. However, when the lipopolysaccharide layer is disrupted (by ethylenediamine tetraacetic acid, detergents, etc.) cells immediately become sensitive to external murein hydrolases.

The lysis systems of some ‘small genome’ viruses are different. Phages Qβ and ØX174 do not employ endolysins in the classical sense, but produce single proteins that interfere with murein biosynthesis by blocking either MraY or MurA, which are essential catalysts of murein biosynthesis and assembly [8, 9]. Thus, they have been termed protein-antibiotics, and might have a future as such.

This review presents an overview on the latest developments in the field on endolysin research, with respect to both fundamental questions and the various applications of these interesting enzymes.

Section snippets

Modular design of endolysin — form follows function

Endolysins must perform two basic functions: substrate recognition and enzymatic hydrolysis. This is achieved by the combination of distinct polypeptide modules that are dedicated these roles. Most of the endolysins studied to date belong to category 1 of the molecular catalysts that are present in nature [10]. They are composed of at least two clearly separated functional domains: the N-terminal domain(s) generally harbor the enzymatic activity, whereas the cell wall binding domains (CBDs)

Enzymatically active domains

The catalytic modules provide two different basic enzymatic activities: glycosidases hydrolyze the linkages of the aminosugar moieties, and amidases and peptidases attack the amide or peptide bonds of the cross-linking peptide stems and interpeptide bridges (Figure 1b). Of all endolysins reported to date, the majority appear to be amidases and muramidases. The l-alanoyl-d-glutamate peptidase activity has only been found in Listeria phage, and several of the interpeptide bridge-specific

Cell wall binding domains

Through evolution, endolysins appear to have acquired rather stringent substrate specificities. At least in some cases, this can be linked to the presence of CBDs. Therefore, specificity seems to be based upon selective targeting of the enzyme to its potential substrate. The N-terminal enzymatic domain cannot be active without being in proximity to its substrate, simply because it does not have sufficient substrate affinity by itself. Instead, recent evidence has indicated that this specific

Endolysin crystal structures

The three-dimensional crystal structures of only a few true endolysins are available; many of the enzymes do not appear to be suitable for the formation of crystals. The most prominent examples are the T4 lysozyme, a well studied prototype muramidase that is extensively used for all sorts of molecular and crystallographic studies [18], and the T7 amidase, which assumes a dual function in cell lysis and in inhibition of RNA polymerase [19]. As a step towards the elucidation of the nature of the

Current and future applications

The bonds that are targeted and hydrolyzed by endolysins are only present in bacterial cell walls. Although endolysins are designed to work from within infected cells, they work equally well when applied exogenously to Gram-positive cells. Often, a minute amount of purified recombinant enzyme is sufficient to rapidly lyse a dense suspension of cells within minutes or even seconds (Figure 3). Owing to the unusual substrate specificity and the high activity of endolysins, they have been applied

Conclusions

Over the past years, we have learned that there is much more to phage endolysins than previously thought, not only in terms of diversity but also with respect to target specificity, (self)regulation, and interaction with other cellular systems. As bacteriophage are the most abundant self-replicating units on earth, and the majority of phage are tailed-phages and therefore feature an endolysin of some sort, they appear to represent an enormous reservoir of tools potentially useful for the many

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

I wish to thank all former and present laboratory members who over the years contributed with their projects to the research on phage and their endolysins, Ingo Korndoerfer, Mark Turner and Jan Kretzer for sharing unpublished data, and Mathias Schmelcher for help in preparing Figure 3. Thanks are also due to Steven Hagens and Kwang-Pyo Kim for critical reading of the manuscript. Last, but not least, I am grateful to Melanie, for tolerating long daily working hours and a few weekends in the

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