Guards of the great wall: bacterial lysozyme inhibitors Lien Callewaert 1 , Joris M. Van Herreweghe 1 , Lise Vanderkelen 1 , Seppe Leysen 2 , Arnout Voet 3 and Chris W. Michiels 1 1 Laboratory of Food Microbiology, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium 2 Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49 bus 822, 3000 Leuven, Belgium 3 Laboratory for Biomolecular Modelling and BioMacS, KU Leuven, Celestijnenlaan 200G bus 2403, 3001 Leuven, Belgium Peptidoglycan is the major structural component of the bacterial cell wall. It provides resistance against turgor and its cleavage by hydrolases such as lysozymes results in bacteriolysis. Most, if not all, animals produce lyso- zymes as key effectors of their innate immune system. Recently, highly specific bacterial proteinaceous lyso- zyme inhibitors against the three major animal lysozyme families have been discovered in bacteria, and these may represent a bacterial answer to animal lysozymes. Here, we will review their properties and phylogenetic distri- bution, present their structure and molecular interaction mechanism with lysozyme, and discuss their possible biological functions and potential applications. Bacterial lysozyme inhibitors: a versatile answer to a versatile enzyme Peptidoglycan (PG) is an essential and unique cross-linked cell wall heteropolymer in bacteria that forms a sacculus around the entire cell. Its major function is to provide resistance against turgor pressure and, consequently, hy- drolysis of PG by bacterial cell-wall hydrolases (BCWHs) renders bacteria sensitive to lysis. BCWHs display a wide structural and functional diversity, and include enzymes that split the b-(1,4) glycosidic bond between N-acetyl- muramic acid (NAM) and N-acetylglucosamine (NAG) in the PG backbone (N-acetyl-b-D-muramidases or lysozymes, lytic transglycosylases (LTs) and N-acetyl-b-D-glucosa- minidases), and enzymes that split the peptide cross-links between the NAM residues (various amidases and endo- peptidases) [1]. These enzymes are found in organisms from all major taxa where they have diverse biological functions such as antibacterial defense (plants, animals) [2,3], the digestion of bacteria as food (animals, protozoa) [2,4], cell-wall synthesis and turnover (bacteria) [5,6], resuscita- tion from dormancy (bacteria) [7], and penetration and lysis of bacteria (bacterial viruses) [8]. Probably the best-studied group of BCWHs are the lysozymes. They are major players in the innate immune system of animals by virtue of their capacity to kill pathogenic bacteria, but also because their action generates PG fragments that are recognized by a variety of immune receptors and thereby trigger an immune response (Box 1). Within the animal kingdom, three main types of lysozymes are distinguished: c- (conventional or chicken), g- (goose), and i- (invertebrate) type lysozyme. Whereas c- and g-type lysozymes, or at least the genes encoding them, are present in all vertebrates, invertebrates typically produce i-type, and sometimes also c-type (e.g., Arthropoda) or g-type (e.g., Mollusca) lysozymes [2,4]. In view of the role of lysozyme in antibacterial protection in animals, it is not surprising that bacteria in turn have evolved mechanisms to evade PG hydrolysis. Chemical modifications that make PG lysozyme-resistant are well- documented (Box 2). In addition, a series of recent papers show that some bacteria produce highly specific and potent proteinaceous lysozyme inhibitors. These inhibitors are the first of their kind, and the structure and mode of interaction of several inhibitors with their cognate lysozyme has been elucidated. Lysozyme inhibitors effectively protect bacteria against lysozyme challenge and thus could contribute to host colonization or infection. Furthermore, they can be antici- pated to modulate the host immune response by interfering with the release of PG fragments during infection. Finally, some findings suggest a role in the regulation of bacterial PG metabolism or in providing immunity against a lysozyme involved in bacterial competition. As such, bacterial lyso- zyme inhibitors offer interesting perspectives for the development of novel antibacterial and anti-inflammatory agents. Screening has thus far focused almost exclusively on inhibitors of animal lysozymes, and although there are some sporadic examples of bacterial inhibitors of other BCWHs, little is known about them. Therefore, we will focus here on bacterial lysozyme inhibitors and review their properties and phylogenetic distribution, present their structure and molecular basis for interaction with lysozyme, and finally discuss their (possible) biological functions and potential applications. Properties and phylogenetic distribution of bacterial lysozyme inhibitors The first bacterial lysozyme inhibitor (Ivy, inhibitor of vertebrate lysozyme) was discovered by serendipity in Escherichia coli [9], and specifically inhibits c-type lyso- zyme. A few years later, a new group of c-type lysozyme inhibitors was discovered and designated the MliC/PliC Review Corresponding author: Michiels, C.W. (chris.michiels@biw.kuleuven.be). Keywords: bacterial lysozyme inhibitors; bacteria–host interaction; peptidoglycan; autolysin inhibition. 0966-842X/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2012.06.005 Trends in Microbiology, October 2012, Vol. 20, No. 10 501