Where is lysozyme made

Our blood is the worst place to have bacteria grow, as they would be delivered to all corners of the body. In the blood, lysozyme provides some protection, along with the more powerful methods employed by the immune system.

Lysozyme left and a form with an engineered disulfide linkage right. Lysozyme is a small, stable enzyme, making ideal for research into protein structure and function. Brian Matthews at the University of Oregon has performed a remarkable series of experiments, using lysozyme as the laboratory for study. He has performed hundreds of mutations on the lysozyme molecule made by a bacteriophage, changing one or more amino acids in the protein chain to a different one.

He has studied the effect of removing large residues inside the protein, leaving a hole, or cramming a large amino acid inside, where it would not normally fit. He has attempted to create new active sites by creating new molecule-shaped pockets.

Structures of hundreds of these mutant lysozymes are available at the PDB--so many, in fact, that lysozyme is the most common protein in the PDB. The example shown here is a mutant were two amino acids shown in green have been changed to cysteine, forming a new disulfide bridge the two bright yellow atoms in the mutant. References R. Kuroki, L. Matthews A covalent enzyme-substrate intermediate with saccharide distortion in a mutant T4 lysozyme.

Science , Pjura, M. For instance, we recently reported that 2 PG-recycling enzymes, the lytic transglycosylases LtgA and LtgD, are important for lysozyme resistance in N. The related cell wall—recycling homologs in Escherichia coli similarly contribute to envelope integrity and lysozyme resistance [ 70 ]. This observation suggests that inhibiting lytic transglycosylase activity, for instance through the antibiotic bulgecin A [ 71 ], could effectively combat infections with gram-negative bacteria by reducing envelope integrity and consequently enhancing their sensitivity to killing by lysozyme and potentially other innate immune components.

Cationic antimicrobial peptides such as lactoferrin synergize with lysozyme for the enhanced killing of gram-negative bacteria through a proposed mechanism by which lactoferrin permeabilizes the outer membrane to enhance the access of lysozyme to periplasmic PG [ 25 ].

Lysozyme itself can form pores on bacterial membranes in some contexts [ 8 , 9 ], yet it is still unclear if these pores are sufficient to enhance the transit of other lysozyme molecules to the periplasm to enzymatically degrade PG.

Some gram-negative bacteria, such as Pseudomonas aeruginosa and E. Instead, they express a periplasmic protein inhibitor of lysozyme that is termed Ivy [ 73 — 75 ]. Inhibition occurs through a loop protrusion in Ivy that occludes the active site of lysozyme via a lock-and-key mechanism [ 75 ]. Ivy is important for in vitro resistance to lysozyme for E. Furthermore, Ivy is important for the survival of Y.

Other bacteria produce additional periplasmic lysozyme inhibitors such as MliC and PliC reviewed in [ 77 ]. In addition to inhibiting lysozyme, Ivy-type proteins also inhibit bacterial lytic transglycosylases [ 73 ].

As described above, lytic transglycosylase—mediated remodeling of the cell wall optimizes envelope integrity and contributes to the defense against lysozyme.

However, unrestrained lytic transglycosylase activity can reduce PG tensile strength and lead to autolysis. Thus, Ivy-type inhibitors play complex and overlapping roles in gram-negative bacterial physiology, immune modulation, and host defense, and most studies have not discriminated among these functions.

For instance, Legionella pneumophila EnhC inhibits the lytic transglycosylase SltL and enhances bacterial survival in association with macrophages [ 79 ]. Although bacterially encoded inhibitors of lytic transglycosylases have not been described for gram-positive bacteria, B.

While lysozyme resistance factors appropriately tailor the bacterial response to immune pressure, modifications to PG that increase resistance to lysozyme may have a fitness cost, as reported for S. Thus, it is not surprising that the expression of many of the factors described above is increased upon the exposure to lysozyme or to immune cells [ 14 , 16 , 18 , 81 ]. For example, E. Regulation of lysozyme resistance factors occurs both transcriptionally and posttranscriptionally.

Despite possessing oatA and dltA , E. The 2-component signal transduction system GraRS induces the expression of the dlt operon in S. GraRS is activated by specific cationic antimicrobial peptides, but the mechanism underlying this activation still remains to be elucidated [ 86 ].

Similarly, the 2-component system VirRS in L. Similarly, SpxB in L. Given the number and diversity of lysozyme resistance factors in both gram-negative and gram-positive bacteria, much remains to be learned about how these factors are regulated. Because many nonpathogenic bacteria encode homologs of Ivy, PgdA, and Oat and Pat O- acetyltransferases [ 32 , 44 , 49 , 53 , 74 , 82 , 90 ], we speculate that pathogenic bacteria produce higher levels of lysozyme resistance factors or exert tighter control over the regulation of these factors than commensals, although these comparisons remain to be made.

Future studies should aim to identify the factors involved with regulating lysozyme resistance genes and to characterize their mechanisms of regulation. Many studies testing the contribution of lysozyme to immune cell responses have relied upon mice that lack lysozyme M gene, lysM.

Lysozyme M is homologous to the single human lysozyme and is produced by phagocytes and other myeloid cells [ 95 ]. Mice also produce a second lysozyme, lysozyme P, which is expressed by intestinal Paneth cells. Lysozyme produced by neutrophils and macrophages can be delivered to bacterium-containing phagosomes [ 1 ]. Accordingly, bacteria that are more sensitive to lysozyme are more likely to be degraded in the phagosomes of macrophages in a LysM-dependent manner [ 31 , 96 ].

In human neutrophils, we recently demonstrated a correlation between the susceptibility of N. The following sections will cover these inflammatory responses mainly in the context of phagocytes.

PG is made by almost all bacteria but not by eukaryotes, making it an excellent target for pattern recognition receptors. Notably, sufficient quantities of stimulatory PG can be released by lysozyme even when lysozyme does not markedly affect bacterial viability [ 96 ].

To date, no modifications that affect the ability of lysozyme to hydrolyze the glycan backbone of PG are implicated in signaling via NOD1; however, NOD1 recognition is reduced by alterations in the PG peptide stem that also affect susceptibility to lysozyme, such as N- myristoylation or the amidation of glutamic acid [ 97 , ].

In contrast, the addition of a stearoyl fatty acid to the C6 distal O -acetyl group in NAM does not inhibit NOD2 signaling and in fact enhances it by allowing for the direct cytosolic entry of MDP [ ]. Chemically synthesized PG moieties of differing glycan lengths e. The cell wall of L. The cell wall from the L. NOD1 is broadly expressed in a variety of cell types, including epithelial cells, and thus contributes to pro-inflammatory signaling in these cell types [ 98 ].

The expression of NOD1 is relatively low in phagocytes, but NOD1 has been implicated in altering phagocyte function in vivo, although it is still unclear whether this is driven by a phagocyte-specific NOD1 response [ — ]. In contrast, NOD2 expression is largely restricted to phagocytes and some specialized cell types, such as intestinal Paneth cells [ 98 , — ].

In phagocytes, the current working model for the activation of NOD family receptors posits that bacteria are phagocytosed and directed into lysosomes containing lysozyme and other antimicrobial components. There, intact, insoluble PG is processed into PG fragments in a lysozyme-dependent manner. PG monomers are then transported across the endosomal membrane via SLC15 family peptide transporters to NOD proteins, which dock on the cytosolic face of the endosome [ 96 , , ].

Phagocytes appear to be optimized to respond to phagosomally produced PG fragments, not extracellular ones, because peripheral monocytes and neutrophils are poorly responsive to extracellular MDP [ ], and macrophages only macropinocytose soluble MDP at high concentrations of ligand [ ]. Further testing of this model has proven challenging because primary phagocytes are poorly genetically manipulable and bear limited resemblance to the favored model for NOD biology, the immortalized HEK cell line in which NOD proteins are overexpressed.

Unlike phagocytes, HEK cells can detect exogenous, soluble PG, which bypasses the need for phagosomal processing [ , ]. Taken together, these data show that the ability of lysozyme to digest PG alters the production of ligands that are recognized by NODs.

Two of the remaining outstanding questions in this field that are germane to this review include defining what structures of PG are ultimately recognized by NODs in phagocytes and how modifications that alter lysozyme-mediated processing manipulate that recognition. The lysozyme-mediated degradation of bacteria enhances the release of immunomodulatory bacterial products, including but not limited to PG.

For example, lysozyme-sensitive S. Similarly, lysozyme-sensitive L. When lysozyme in macrophages is inhibited by using exogenously added NAG polymer i. One direct mechanism for inflammasome activation by PG was recently elucidated by Wolf et al. N -deacetylation of NAG abrogates this response, linking PG modifications and, by extension, susceptibility to lysozyme-mediated degradation to NLRP3 activation [ ]. Inflammasome activation may also be indirect through the lysozyme-catalyzed release of other stimulatory bacterial factors.

At the site of infection, extracellular lysozyme red sector , which is secreted locally by the epithelium, can kill bacteria, leading to the release of PAMPs, including but not limited to monomeric PG. This can initiate an epithelial-driven response that leads to phagocyte recruitment not depicted here. Resident or recruited macrophages also secrete lysozyme extracellularly and can internalize bacteria, delivering lysozyme to the bacterium-containing phagosome.

In macrophages, bacterial degradation by phagosomal lysozyme releases PAMPs that stimulate a robust proinflammatory cytokine response and activate the inflammasome. Neutrophil activities may be similarly enhanced by lysozyme-mediated degradation of phagosomal bacteria, akin to macrophages. Because phagocytes poorly respond to extracellular, monomeric PG and monomeric PG cannot activate complement, the degradation of bacterial PG by extracellular lysozyme serves to restrict phagocyte activation and recruitment.

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Meat Animal Research Center, P. You can also search for this author in PubMed Google Scholar. Correspondence to W. WO and JW contributed to the writing of this review paper.

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