Deception point: Peptidoglycan modification as a means of immune evasion

The ability to evade host immune surveillance is a critical virulence determinant for any pathogenic microorganism. The intracellular pathogen Listeria monocytogenes is highly adept at reaching privileged niches within the host without alerting immune responses. Upon infection, Listeria penetrates epithelial cells in the gastrointestinal tract and spreads silently from cell to cell. Once this barrier is breached, it has the ability to escape the phagosome and replicate within phagocytic cells, using them as vehicles to disseminate throughout the host (reviewed in ref. 1). The capacity to defend against the onslaught of immune defenses is another key aspect to being a successful pathogen. In this issue of PNAS, Boneca et al. (2) demonstrate that Listeria modifies peptidoglycan (PG) in its cell wall, thereby conferring resistance to the host bacteriolytic product, lysozyme. By preventing bacterial degradation and subsequent release of immunostimulants, PG modification is therefore also a means by which Listeria evades immune detection by host pattern–recognition receptors (PRRs). Importantly, although many pathogens are known to modify their PG, this report by Boneca et al. (2) is the first to demonstrate that PG N-deacetylation has an important role in virulence and evasion of host defense.

PG is a large polymer that provides much of the strength and rigidity to bacterial cell walls (reviewed in ref. 3). It consists of long glycan chains of alternating β-1,4-linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) subunits, which are cross-linked via flexible peptide bridges. Boneca et al. (2) analyzed the muropeptide composition of L. monocytogenes to reveal that ≈50% of its PG is N-deacetylated. PG modifications including amidation, acetylation, and glycolation are used by many Gram-positive and -negative pathogens and have been shown to mediate bacterial resistance to lysozyme (4). O-acetylation of PG is best studied in Staphylococcus aureus, where up to 90% of PG residues present are modified by the acetylase OatA, resulting in substantial resistance to the muramidase activity of lysozyme (5). Lysozyme resistance also occurs in mycobacterial species where NAM and NAG are N-glycosylated by NamH (6).

Lysozyme is a PG N-acetyl-muramoylhydrolase that is found in animals, plants, insects, viruses, and bacteria (4). Because lysozyme is cationic, it closely adheres to bacteria through electrostatic interactions with negatively charged teichoic and lipoteichoic acids and phospholipids on the bacterial surface. This interaction can result in bacterial lysis by (i) autolysins, which are activated upon displacement of cell-surface divalent cations by lysozyme, or (ii) the enzymatic properties of lysozyme that hydrolyze β-glycosolic bonds between NAG and NAM (4). To investigate the importance of PG N-deacetylation to Listeria's ability to withstand lysozyme, Boneca et al. (2) identified the PG N-deacetylase gene, pgdA, which was essential for N-deacetylation of Listeria's PG. Consistent with previous findings, the authors show that the pgdA mutant was significantly more sensitive to killing by lysozyme. Increasing evidence suggests that the nonenzymatic function of lysozyme is particularly critical for its bactericidal activity against both Gram-positive and -negative organisms in vivo (7, 8). Therefore, it would be interesting to determine whether PG N-deacetylation by Listeria protects it from the enzymatic or cationic function of lysozyme. Macrophages are likely key immune cells in the defense against Listeria infection and are abundant producers of lysozyme. Accordingly, this study also found that, if Listeria cannot N-deacetylate its PG (ΔpgdA), it is severely impaired in its ability to survive and/or replicate within macrophages. Interestingly, the pgdA mutant does not escape the phagosome, leading the authors to speculate that bacteria are killed before they are able to escape.

PRRs such as Toll-like receptors (TLRs) and nuclear oligomerization domain (Nod) proteins recognize pathogen-associated molecular patterns (PAMPs) found on the outer surface of bacteria. Ligation of PRRs with their respective PAMPs initiates NF-κB signaling, which results in the production of various chemokines, growth factors, antimicrobial peptides, proinflammatory cytokines, and other products required for clearance of the infection. PG is a crucial bacterial PAMP sensed intracellularly by Nod1 and Nod2 (reviewed in ref. 9). In the present study (2), they investigated the role of Listeria PG N-deacetylation on immune detection and the inflammatory responses of macrophages. As shown previously, PG purified from wild-type (WT) Listeria poorly activates NF-κB (10). In comparison, fully acetylated PG purified from the pgdA mutant, or WT PG predigested with a muramidase, increased NF-κB activation in a Nod1- and Nod2-dependent manner. These data have forced a reinterpretation of past studies where the inability of certain PGs to stimulate NF-κB was rationalized by claiming that they were inaccessible to intracellular Nod proteins (10).

Next, the consequence of PG N-deacetylation on NF-κB induction was explored by analyzing the inflammatory cytokine production by macrophages. The pgdA mutant stimulated much higher levels of proinflammatory cytokines when compared with WT bacteria, and this enhanced cytokine production was primarily dependent on TLR2 and Nod1. Nod1 (11), Nod2 (12), and TLR2 (13) are important for innate immune recognition of Listeria, yet this report describes a mechanism by which Listeria evades detection by these receptors. Hopefully, future studies will shed light on this apparent dichotomy. Together, however, Boneca et al.'s (2) data suggest that WT PG has the ability to provoke NF-κB activation, but by preventing PG degradation by muramidases like lysozyme, N-deacetylation prevents immune detection and subsequent NF-κB activation, influencing immune detection and modulating responses to bacterial infection.

The capacity to defend against immune defenses is key to being a successful pathogen.

To investigate the role of PG N-deacetylation in virulence, Boneca et al. (2) used oral inoculation of human E-cadherin transgenic mice (14) to model the early stages of infection and i.v. inoculation of mice to model the later, systemic stages of infection. In vivo challenge revealed that the Listeria pgdA mutant was severely attenuated in both the intestinal lumen and in systemic organs like the spleen and liver. The authors speculate that the susceptibility of the pgdA mutant to lysozyme and other antimicrobial agents produced by Paneth cells in the small intestine could account for the rapid clearance from the lumen (Fig. 1A). At systemic sites, they propose that bacterial degradation by phagocyte lysozyme, and the resultant immune stimulation, permits efficient clearance of infection (Fig. 1B and C). In addition, the inability of the pgdA mutant to escape the phagosome could have a significant effect on the extent of systemic infection. Lysozyme's importance in innate defenses is underscored by in vitro work showing numerous bacteria to be susceptible to lysosomal degradation (4) and in vivo work showing that transgenic mice deficient in lysozyme have increased susceptibility and prolonged inflammatory responses to Gram-positive pathogens (15). The specific role of lysozyme in clearance of the Listeria at systemic sites could be readily addressed by i.v. infecting lysozyme knockout mice (16).

Fig. 1.

Model of host detection and destruction of Listeria in the absence of PG N-deacetylation. (A) Listeria pgdA mutants are degraded by lysozyme in the intestinal lumen. PG muropeptides released upon degradation may be delivered into the host cell cytoplasm by endogenous transporters whereby they would stimulate Nod-dependent epithelial inflammatory responses (17). (B) Listeria pgdA mutants are taken up into macrophage phagosomes, degraded by phagosomal lysozyme, and release lipoteichoic acid that is sensed by phagosomal TLR2 and PG murpeptides that can be transported out of the phagosome and detected in the cytosol by Nod proteins. Ligation of these PRRs induces an NF-κB-dependent inflammatory response by the macrophage. (C) Listeria pgdA mutants cannot escape macrophage phagosomes and therefore are unable to efficiently spread within systemic sites.

Boneca et al. (2) highlight how PG modification in Listeria is an immune evasion mechanism that protects the bacteria at every stage of infection, from resistance to host antimicrobial agents in the gastrointestinal tract, to survival in professional phagocytes, to its ability to modulate inflammatory responses in the host. These findings are important because PG modification is likely used by many pathogens as a means of immune evasion.