The precise role of lysozyme during innate immunity to microbial infection has yet to be fully appreciated. The aim of this study was to assess the role of host lysozyme resistance by comparing L. monocytogenes strains that were either resistant or sensitive to lysozyme. The results of this study showed that lysozyme acts extracellularly, within phagocytic vacuoles, and surprisingly in the cytosol of infected cells. In phagosomes, lysozyme activity led to the release of bacterial ligands that activated a vacuole-specific program of cytokine induction. In the cytosol, lysozyme led to bacteriolysis, thereby activating two distinct cytosolic innate immune pathways, one leading to the expression of IFN-β and another leading to DNA-dependent, AIM2-dependent pyroptosis. All of the phenotypes associated with lysozyme sensitivity were reversed during infection of macrophages lacking LysM. Surprisingly, all of the above-mentioned phenotypes were restored by simply adding lysozyme to the extracellular medium.
Transcription of IL-12 and IL-1β is stimulated by L. monocytogenes
trapped in phagosomes (43
). Lysozyme-sensitive L. monocytogenes
stimulated increased expression of these cytokines in LLO−
and LLO-expressing backgrounds. TLR2 is a candidate receptor involved in this pathway since it is both localized at the cell surface and recruited to phagosomes upon internalization of microbes (44
). TLR2 has been reported to detect various cell wall-associated ligands, including peptidoglycan (although there is controversy over whether it is a bona fide ligand), and has been shown to form heterodimers with TLR1 or TLR6 to detect lipopeptides and lipoteichoic acid (LTA) (29
). It is likely that lysozyme activity generates increased cell wall fragments that are detected by TLR2. However, although vacuolar cytokines were dependent on MyD88, infection with Pgd− L. monocytogenes
induced similar levels of IL-1β and IL-12 in TLR2−
and wild-type BMM (data not shown). Therefore, the precise nature of the bacterial ligand(s) or host receptor(s) is not known.
Cytosolic L. monocytogenes
strains induce a MyD88-independent response that is monitored by IFN-β expression. Here, IFN-β expression is elevated approximately 2-fold by lysozyme-sensitive mutants. One possible explanation is that cytosolic bacteria are lysed, resulting in the release of DNA and cyclic di-AMP, two ligands that activate IFN-β expression (31
). A second possibility is that N-deacetylated peptidoglycan shed from growing bacteria has enhanced stimulatory activity. Indeed, Boneca et al. reported that N-deacetylated peptidoglycan had increased NOD1 stimulatory activity on peritoneal elicited macrophages and that digested Pgd−
peptidoglycan induced increased NF-κB activity in both a NOD1- and NOD2-dependent manner (3
). However, using NOD1−
, or NOD1−
BMM, we still observed an approximate 3-fold difference in IFN-β expression levels between wild-type and Pgd−
bacteria (data not shown). While it is possible that NOD1 and NOD2 expression levels were different, our data favor the hypothesis that increased bacteriolysis and release of nucleic acids led to enhanced IFN-β expression.
This study also revealed that lysozyme acts in the cell cytosol, causing bacteriolysis and subsequent host cell death. These results support the recently emerging concept that cytosolic bacteriolysis is linked to activation of the AIM2-dependent inflammasome (51
). Indeed, using strains differentially susceptible to lysozyme, we showed that increased lysozyme sensitivity correlates with increased bacteriolysis and AIM2-dependent pyroptosis. This is the first study to show a link between lysozyme and AIM2 stimulation. However, a previous study has linked lysozyme activity to the NLRP3 inflammasome. Shimada et al. have shown that vacuolar digestion of S. aureus
peptidoglycan by lysozyme is required for NLRP3 stimulation (54
). However, we found no significant role for NLRP3 in response to lysozyme-sensitive L. monocytogenes
strains despite distinct lysozyme activity in the vacuole, suggesting that L. monocytogenes
peptidoglycan is not sensed via the same pathway. It is unknown whether S. aureus
lysozyme-digested peptidoglycan is the ligand detected directly by NLRP3 or whether activation is stimulated by its feature as a small crystalline molecule (30
). Nevertheless, similar to S. aureus
, L. monocytogenes
specifically subverts inflammasome activation by modification of its peptidoglycan.
All of the phenotypes associated with lysozyme sensitivity could be restored simply by adding lysozyme to the culture medium. In our experiment, we used 1 mg/ml hen egg white lysozyme (HEWL), which is within the biological range (6
). Although HEWL has the same enzymatic activity as mammalian lysozyme, it may have properties that differ in specificity for deacetylated peptidoglycan or access to BMM cytosol compared to endogenous mouse lysozyme M. There is evidence that macrophages internalize other extracellular peptides in addition to lysozyme. For example, Tan et al. have reported that macrophages can acquire antimicrobial peptide defensins from apoptotic neutrophil granules to become increasingly resistant to Mycobacterium tuberculosis
). The Lehrer lab has shown that macrophages can take up alpha-defensins (23
; R. I. Lehrer, personal communication). It has also been shown that anti-LLO antibodies can act in a phagosome to inhibit L. monocytogenes
). Therefore, soluble innate immune factors, including secreted lysozyme, may also concentrate in macrophages.
How lysozyme enters the cytosol is less obvious. However, several other small cationic peptides have been reported to translocate from the extracellular milieu to the cytosol. HIV-1 Tat, herpes simplex virus 1 VP22 transcription factor, and a Drosophila
protein, Antp, have been shown to enter cells from the culture medium and localize in the cytoplasm. Of these translocated proteins, HIV-1 Tat has been the best studied. Tat is secreted from infected cells and subsequently enters adjacent cells and accumulates in the cytosol (16
). Tat is a 101-amino-acid cationic peptide with a calculated isoelectric point of 9.61 and has been used as a fusion protein to deliver ovalbumin, β-galactosidase, peroxidase, and green fluorescent protein into cells (14
). A basic domain rich in arginine and lysine residues has been identified in Tat as the region required for translocation (41
). Although lysozyme does not share a homologous domain, it too is a small cationic peptide of 14 kDa, with a calculated isoelectric point of 9.11. Although speculative, its similar features in size and cationic quality are properties that could suggest a mechanism for its access to the cytosol.
Lysozyme-sensitive L. monocytogenes
strains were severely attenuated in the mouse model of listeriosis. However, the loss of virulence was not rescued in LysM−
mice. The most likely explanation was a compensatory and redundant mechanism of LysP. LysP compensation in LysM−
mice has been reported separately by Ganz et al. and Markart et al. (17
). Markart et al. showed that LysP mRNA is typically not expressed in the alveolar space in wild-type mice but is upregulated significantly in LysM−
). Ganz et al. showed that lysozyme was detected with an antibody that recognized both M and P isoforms in alveolar and peritoneal macrophages (17
). Certainly, LysP compensatory expression in relative tissues could explain the lack of rescue. Lysozyme P circulating in the blood may also be a contributing factor. Pgd−
Oat− L. monocytogenes
had a log decrease in CFU in liver by 30 min (data not shown), suggesting an early encounter with lysozyme. Also, based on our results, circulating lysozyme P may enter macrophages and act intracellularly in a vacuole and the host cell cytosol.
In addition, lysozyme-sensitive L. monocytogenes
may be susceptible to other host cell hydrolytic enzymes or stresses not present in BMM. Popowska et al. have shown that Pgd− L. monocytogenes
is more sensitive to autolysis-inducing agents, Triton X-100 and EDTA, and various antibiotics (46
). Indeed, it is predicted that Pgd− L. monocytogenes
bearing N-acetylated glucosamine residues would have a more negative surface charge due to lack of an exposed primary amine residue and would thus be more susceptible to cationic peptides (63
). However, Pgd−
Oat− L. monocytogenes
strains were not sensitive to cationic peptides, including LP9, a human lysozyme-derived 9-mer, in comparison to a strain lacking MprF, which is known to be sensitive to cationic agents (see Fig. S2 in the supplemental material) (24
). In the future, it will be of interest to determine whether it is the lytic activity of mouse lysozyme or its cationic properties that act on Pgd−
Oat− L. monocytogenes
We conclude that L. monocytogenes
resistance to lysozyme is an essential determinant of pathogenesis. Indeed, many bacterial pathogens are lysozyme resistant (12
). The role of lysozyme appears to be multifaceted. We and others have shown that lysozyme activity can kill bacteria and stimulate inflammation, but in other contexts, it has been shown to inhibit inflammation. Ganz et al. showed that Micrococcus luteus
induced increased inflammation in LysM−
mice, suggesting that peptidoglycan degradation by lysozyme is required to downregulate inflammation (17
). Thus, one role of lysozyme may be to degrade potentially proinflammatory peptidoglycan fragments associated with commensal or nonpathogenic bacteria, which may be a strategy to avoid mounting an inappropriate inflammatory response. This may be similar to a mechanism whereby host enzymes inactivate bacterial lipopolysaccharide to avoid prolonged tolerance and increased inflammation (39
). However, lysozyme also serves to generate peptidoglycan fragments that are important immunostimulatory ligands during infection. Davis and Weiser suggest that modified peptidoglycan that is more resistant to hydrolysis by lysozyme results in the generation of larger fragments, which might be differentially detected by a host (12
). The ability of lysozyme to act from within cells (in both phagosomes and the cytosol) and extracellularly highlights its role as a versatile antimicrobial factor that contributes to multiple aspects of host defense during bacterial infection.