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The Journal of Immunology, 2006, 177: 519-526.
Copyright © 2006 by The American Association of Immunologists

The Peptidoglycan-Degrading Property of Lysozyme Is Not Required for Bactericidal Activity In Vivo1

James A. Nash, Tiffany Nicole S. Ballard, Timothy E. Weaver and Henry T. Akinbi2

Division of Pulmonary Biology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysMD53S) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysMD53S in the respiratory epithelium of wild-type (LysM+/+/LysMD53S) or lysozyme Mnull mice (LysM–/–/LysMD53S) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM–/– mice that was completely reversed by targeted expression of LysMD53S. These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rapid elimination of inhaled microorganisms from the airways and distal lung airspaces is essential to prevent colonization and persistent inflammation that could compromise gas exchange. Antimicrobial proteins/peptides play a key role in promoting a sterile gas exchange surface by directly killing and/or facilitating phagocytosis of microorganisms by resident lung macrophages. Surfactant protein (SP)3 A and SP-D are relatively abundant host defense proteins that play an important role in opsonizing, aggregating, and enhancing clearance of microbes by alveolar macrophages (1). SP-A and SP-D may also directly kill Gram-negative bacteria (2); however, the majority of bactericidal activity in the respiratory passages appears to be contributed by lysozyme, lactoferrin, and secretory leukoprotease inhibitor (3, 4).

In the human and ferret lung, lysozyme is secreted predominantly by serous cells of the submucosal glands with a smaller contribution by airway epithelial cells (5, 6). Immunodepletion of lysozyme decreased bactericidal activity in human airway secretions by ~50%, suggesting that lysozyme derived from submucosal glands is a major component of airway host defense (4). Mice and rats have relatively few submucosal glands but secrete lysozyme with pulmonary surfactant from alveolar type II epithelial cells (7, 8); in addition, macrophages also secrete lysozyme into the alveolar airspaces (9). Although the relative contribution of macrophages and type II cells to alveolar lysozyme content is not known, lysozyme accounts for as much as 6–7% of soluble protein in rat bronchoalveolar lavage fluid (8). Elevated concentration of lysozyme in the alveolar airspaces of transgenic mice conferred resistance to infection by Pseudomonas aeruginosa or group B Streptococcus and significantly enhanced survival (10); in contrast, lysozyme deficiency increased bacterial burden and mortality following intratracheal infection with Klebsiella pneumoniae (11) or P. aeruginosa (12). Thus, lysozyme likely plays an important role in innate host defense of the lungs.

Mice express two lysozyme genes, LysM and LysP (13, 14, 15). Lysozyme M is the predominant protein in most cells (16), including alveolar macrophages and type II cells. Disruption of the gene encoding LysM (17) leads to up-regulation of LysP resulting in partial compensation of muramidase activity (15, 18). Although recombinant lysozyme M and P were equally effective at killing Gram-positive bacteria in vitro, lysozyme M was somewhat more effective at killing selected Gram-negative bacteria (15). The lower antimicrobial activity of lysozyme P, coupled with incomplete compensation in LysM–/– mice, likely accounts for the increased susceptibility of null mice to bacterial colonization and inflammation (11, 18).

Lysozyme hydrolyzes the bond between N-acetyl glucosamine and N-acetyl muramic acid (muramidase activity) leading to degradation of peptidoglycan in the cell wall of Gram-positive bacteria. The three-dimensional structure of lysozyme M, determined by nuclear magnetic resonance spectroscopy, identified E35 and D53 as active site residues in the mouse enzyme (19). Substitution of serine for aspartic acid in the active site of hen egg white lysozyme completely ablated muramidase activity (20). Interestingly, catalytically inactive chicken lysozyme was as effective as wild-type (WT) lysozyme in killing Staphylococcus aureus, Bacillus subtilis, and Bacillus cereus, suggesting that the ability to degrade peptidoglycan was not essential to kill these Gram-positive bacteria in vitro (20). To determine whether muramidase-deficient lysozyme could confer resistance to infection by Gram-positive or Gram-negative bacteria in vivo, a mouse lysozyme MD53S construct was generated and expressed in type II cells of WT and LysM–/– mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Recombinant WT lysozyme M and D53S protein

Lysozyme M was amplified from mouse type II cell cDNA using specific primers that discriminate between lysozyme M and P. Sequences encoding a polyhistidine tag were added to the 5' end of the downstream primer. Muramidase-deficient lysozyme M (LysMD53S) was generated by site-directed mutagenesis (QuickChange kit; Stratagene) which altered the codon for aspartic acid at position 53 to encode serine. Both LysM and LysMD53S cDNAs were cloned into pVL1393 (BD Pharmingen). Recombinant baculovirus was generated by homologous recombination in Spodoptera frugiperda 9 cells. For expression of recombinant protein, fresh monolayers of 107 Trichoplusia ni cells (Invitrogen Life Technologies) were infected with plaque-purified recombinant virus at a multiplicity of infection of 2 and cultured in serum-free medium for 72 h. Recombinant WT lysozyme M and D53S proteins were purified from the medium by nickel-charged nitriloacetic acid (Ni NTA) chromatography and characterized by Western blotting and silver staining as previously described (15). Alternatively, the LysM and LysMD53S constructs were cloned into pE21-a (EMD Biosciences) for expression in BL21(DES) bacterial cells (EMD Biosciences). Recombinant lysozyme was recovered from bacterial lysates under denaturing conditions, purified by Ni NTA chromatography, and refolded by passage over a buffer exchange column (PD-10; Amersham Biosciences) into 10 mM potassium phosphate (pH 7.4)/10% glycerol. The muramidase activities of WT lysozyme M and D53S proteins were compared as described below (Lysozyme enzyme activity in bronchoalveolar lavage fluid (BALF)). The hexahistidine sequence was not removed from the recombinant proteins as we have previously shown that both muramidase and microbicidal activities are unaffected by the presence of the tag (15).

Mice

The four groups of mice used in the current study included mice deficient in lysozyme M (LysM–/–), transgenic mice expressing the muramidase-deficient lysozyme M transgene (D53S) in the distal respiratory epithelium (LysM+/+/LysMD53S), lysozyme M-deficient mice expressing the D53S transgene in the distal respiratory epithelium (LysM–/–/LysMD53S) and WT mice. Generation and characterization of LysM–/– mice was described previously (11, 17). LysM+/+/LysMD53S transgenic mice were generated by microinjection of fertilized oocytes with a "mammalianized" transgene construct (21) consisting of the 3.7-kb human SP-C promoter, rabbit beta-globin intron I and exon II, a cDNA fragment encoding the D53S transgene and the bovine growth hormone polyadenylation sequence. Transgenic mice were identified by PCR amplification of a fragment spanning the junction of rabbit beta-globin and D53S cDNA from genomic DNA using the following primers: 5'-CCC CTC TGC TAA CGA TGT TC-3' and 5'-AAC AGA TGG CTG GCA ACT AGA-3'. Genotypes of founder mice were confirmed by Southern analyses using a 32P-labeled fragment containing the bovine polyadenylation sequence. LysM+/+/LysMD53S mice were crossed to LysM–/– mice to generate LysM+/–/LysMD53S mice. LysM+/–/LysMD53S siblings were mated to produce LysM–/–/LysMD53S mice. LysM+/+/LysMD53S and LysM–/–/LysMD53S mice were indistinct from WT mice with respect to longevity, fecundity, and somatic growth. All four groups of mice were maintained in the FVB/N genetic background. Mice were handled according to the Institutional Animal Care and Use Committee guidelines at Cincinnati Children’s Hospital Medical Center, and were maintained in a barrier containment facility. Serologies were periodically checked for common murine pathogens. Five- to 7-wk-old mice from each group were used in all experiments.

Analysis of protein expression in BALF

An aliquot of BALF containing 1 µg of protein was analyzed by SDS-PAGE followed by Western blotting with polyclonal rabbit Ab, directed against human lysozyme (Accurate Chemicals and Scientific), as previously described (15). Relative levels of lysozyme proteins in lung BALF were assessed by scanning densitometry using Image-Quant software (Molecular Dynamics).

Spatial expression of lysozyme in the lungs

To assess gross lung structure and expression of lysozyme in the lungs of transgenic mice, lungs from 5-wk-old LysM+/+/LysMD53S transgenic mice and WT littermates (n = 4 for each genotype) were inflation-fixed for immunohistochemistry and light microscopy, as previously described (11). Immunostaining for lysozyme was performed using antiserum directed against human lysozyme (Accurate Chemicals and Scientific). Parallel lung sections were incubated with preimmune rabbit serum to verify the specificity of immunostaining.

Lysozyme enzyme activity assay in BALF

To assess muramidase activity in BALF, an aliquot containing 1 µg of protein was incubated with 1 ml of killed Micrococcus luteus suspended in 0.4 M phosphate buffer (pH 6.7) at an OD of 1 (450 nM) at 37°C. Changes in OD during the 30-min incubation were plotted against time using a recording spectrophotometer. Purified chicken lysozyme was used to generate a standard curve (1 U of enzyme activity = 0.001 change in OD).

Bacteria

GFP-expressing P. aeruginosa, PAOI (provided by Dr. T. Machen, University of California, Berkeley, CA), K. pneumoniae strain K2 (from Dr. Korfhagen, Cincinnati Children’s Hospital, Cincinnati, OH) and a clinical isolate of S. aureus were used in the current study. To minimize variability in virulence, all bacteria were selected from aliquots of the same passage that had been frozen at –70°C in 20% glycerol/PBS. For each experiment, an aliquot of bacteria was thawed and plated on tryptic soy/5% sheep blood agar. A colony was inoculated in 4 ml of Luria Bertani medium (P. aeruginosa, K. pneumoniae) or brain-heart-infusion medium (S. aureus) and grown to late log phase. Bacteria were pelleted from the medium, washed in sterile PBS, and resuspended in 4 ml of HBSS supplemented with 4.5 mM glucose. Bacteria were enumerated as CFU on blood agar plates from serial dilutions in PBS. For each experiment, the inoculum was confirmed by plating dilutions of the aliquot used for intratracheal injection.

Bacterial killing in vitro

One thousand CFU of S. aureus or 1 x 106 CFU of P. aeruginosa were suspended in 100 µl of 10 mM potassium phosphatase (pH 7.4) and incubated for 3 h at 37°C with increasing amounts of purified recombinant lysozyme M, D53S, or buffer alone in quadruplicate in a 96-well plate. Quantitative cultures were performed and viable pathogen counts determined.

Bacterial killing in vivo

The doses of bacteria selected for the current study were based on previous experiments in the FVB/N mouse strain (11, 22). To assess bacterial killing, 1 x 107 CFU P. aeruginosa, 1 x 104 CFU of K. pneumoniae, or 1 x 105 CFU of S. aureus in 100 µl of PBS were administered by intratracheal instillation as previously described (10). Mice were sacrificed 12 h (S. aureus), 24 h (S. aureus, P. aeruginosa, K. pneumonia), or 48 h (S. aureus) postinfection, the lungs were weighed, homogenized, and dilutions were plated for quantitative culture. The numbers of colonies were expressed as CFU per gram of lung tissue. To assess systemic dissemination of infection, splenic homogenates were plated and incubated overnight at 37°C and the number of colonies counted. For each experiment, 8–10 mice from each group were infected. Studies were conducted twice for each pathogen and results pooled.

Lung histopathology

Twenty-four hours following intratracheal challenge with P. aeruginosa, lungs from 5-wk-old LysM–/– mice (n = 4) or LysM–/–/LysMD53S littermates (n = 4) were inflation-fixed and 5-µm paraffin-embedded sections were stained with Brown and Brenn reagents (Poly Scientific). Bacteria were visualized at x250 magnification with a Zeiss microscope.

Survival studies

LysM–/– and LysM–/–/LysMD53S mice (n = 20 for each group) were infected by intratracheal instillation with either 1 x 108 CFU of P. aeruginosa or 1 x 105 CFU of K. pneumoniae suspended in 100 µl of PBS. Water and food were provided ad libitum during the period of observation. The number of surviving mice was documented every 12 h for up to 120 h, at which time surviving mice were sacrificed.

Statistical analyses

Data are expressed as mean ± SEM. For bacterial clearance, data are reported as CFU per gram of lung tissue. Differences between groups were assessed by one-way ANOVA, and differences between means were assessed by contrast comparisons and the Student-Newman-Keuls test (Statview; SAS Institute). Nonparametric survival distributions were estimated to examine differences in survival between LysM–/– and LysM–/–/LysMD53S mice. Differences between the groups were analyzed from the Kaplan-Meier curve by log-rank test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Recombinant WT and mutant lysozyme M

Glutamic acid 35 and aspartic acid 53 are both essential for the muramidase activity of lysozyme M. In the current study, the codon for aspartic acid 53 was mutated to encode serine (D53S). The mutation was confirmed by sequence analysis and cDNA constructs encoding mutant and WT lysozyme M were cloned into baculovirus vectors for expression in insect cells. Recombinant WT and D53S lysozyme, Mr = 14 k, were readily detected and purified from medium, indicating that the D53S substitution did not perturb folding and secretion of mouse lysozyme M (Fig. 1, A and B). To confirm that muramidase activity was ablated in the D53S recombinant protein, lysozyme enzyme activity was assessed using a well-characterized turbidimetric assay. Muramidase activity for the WT protein was 50 U/ng whereas no activity was detected with up to 10-fold more D53S protein (Fig. 1C). Collectively, these results indicate that the D53S substitution completely abrogated muramidase activity without affecting the immunoreactivity or secretion of the protein.


Figure 1
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  		FIGURE 1. Expression and characterization of muramidase-deficient lysozyme. A, cDNAs encoding WT mouse lysozyme M (LysM) or mutant lysozyme M (D53S), in which the codon for aspartic acid (residue 53) was mutated to encode serine, were expressed in S. frugiperda 9 cells. Proteins were purified from the medium by Ni NTA chromatography and assessed by SDS-PAGE and silver staining. All elution fractions (lanes 1–8) were analyzed. B, A total of 0.5 µg of purified recombinant protein was resolved by SDS-PAGE, transferred to nitrocellulose, and probed with Ab that detects both WT and D53S lysozyme M. C, Muramidase activity of recombinant WT (50 ng) or mutant D53S (500 ng) lysozyme M proteins was assessed by turbidimetric assay with M. luteus as substrate. Net absorbance at OD of 450 nm over 25 min of incubation was plotted by a spectrophotometer.

 
The antimicrobial activity of recombinant WT and D53S lysozyme was tested against a Gram-negative bacterium (P. aeruginosa) and a Gram-positive bacterium (S. aureus). The bactericidal activity of muramidase-deficient lysozyme was not significantly different from that of the recombinant WT enzyme (Fig. 2). As previously reported (15), the dose required to kill 50% of P. aeruginosa (70 pM) was lower than that required to kill 50% of S. aureus (110 pM).


Figure 2
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  		FIGURE 2. Bactericidal activity of recombinant WT lysozyme M and D53S. A total of 106 CFU of P. aeruginosa (A) or 103 CFU S. aureus (B) were incubated with 0, 50, 100, or 150 pM of recombinant protein at 37°C for 3 h. The number of CFU was assessed by quantitative culture.

 
Generation and characterization of transgenic mice

The ability of muramidase-deficient lysozyme to kill airway pathogens in vivo was tested by generating mice that expressed the D53S protein in the distal airway epithelium. Five independent LysM+/+/LysMD53S transgenic lines were generated and screened by Western blotting to determine the concentration of lysozyme in BALF (Fig. 3). Lysozyme concentration was elevated ~8-fold in transgenic line 5.7 and this line was subsequently crossed into the lysozyme Mnull background (LysM–/–/LysMD53S). S1 nuclease analyses indicated that the level of lysozyme M mRNA (which included both WT and D53S transcripts) was significantly increased in LysM+/+/LysMD53S transgenic mice compared with WT mice (Fig. 4A). The level of lysozyme M mRNA was similarly elevated in LysM–/–/LysMD53S mice, suggesting that expression of the D53S transgene (rather than increased expression of endogenous (WT) gene) accounted for elevated LysM mRNA in LysM+/+/LysMD53S transgenic mice. Lysozyme P mRNA was detected at relatively low abundance in both WT and LysM+/+/LysMD53S transgenic mice; however, expression of lysozyme P was increased in LysM–/– mice and this increase was maintained when the transgene was expressed in the null background (LysM–/–/LysMD53S).


Figure 3
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  		FIGURE 3. Generation of transgenic mice expressing muramidase-deficient lysozyme. Catalytically inactive lysozyme M (LysMD53S), in which aspartic acid at position 53 was mutated to serine, was expressed under control of the human SP-C promoter in the distal airway epithelium. Five lines of transgenic mice were generated (only four are shown). One microgram of BALF protein from WT or LysM+/+/LysMD53S mice was resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and incubated with lysozyme Ab. The intensity of the lysozyme bands was quantitated by computer-assisted densitometric analysis. Values were normalized to WT; n = 5/group for each transgenic line.

 

Figure 4
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  		FIGURE 4. Characterization of transgenic mice expressing muramidase-deficient lysozyme. A, Total lung RNA (15 µg) from LysM+/+/LysMD53S (lanes 1 and 2), WT (lanes 3 and 4), LysM–/– (lanes 5 and 6), and LysM–/–/LysMD53S (lanes 7 and 8) mice was hybridized overnight with 32P-labeled DNA probes specific for lysozyme M, lysozyme P, or mouse SP-B and subsequently digested with S1 nuclease. The reaction mixture was resolved on a 6% acrylamide gel to identify the 320-bp lysozyme M, 228-bp lysozyme P, and 186-bp SP-B fragments. B, BALF (0.5 µg protein) recovered from 5-wk-old LysM+/+/LysMD53S, WT, LysM–/–, or LysM–/–/LysMD53S mice was fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and detected by Western blotting with lysozyme Ab. C, Cellular localization of lysozyme was assessed by immunohistochemistry on lung sections from 5-wk-old LysM+/+/LysMD53S and WT mice using a rabbit lysozyme Ab. Arrowhead, nonciliated bronchiolar cells; arrows alveolar macrophage. Size bar, 100 µm. D, BALF (1 µg protein) from WT, LysM+/+/LysMD53S, LysM–/–, and LysM–/–/LysMD53S mice was incubated with a suspension of M. luteus. The change in OD was recorded and muramidase activity calculated from a standard curve generated with chicken lysozyme. Muramidase activity was reported as percentage ± SEM of WT.

 
Lysozyme M and P proteins appeared to be present in similar concentrations in BALF of FVB/N mice (Fig. 4B); however, lysozyme P was previously shown to be more immunoreactive than lysozyme M in Western blots, indicating that lysozyme M was in fact the predominant isoform (15). Lysozyme P protein was increased to the same extent in BALF from LysM–/– and LysM–/–/LysMD53S transgenic mice consistent with S1 nuclease analyses (Fig. 4B). The concentration of D53S protein in BALF was similar in LysM+/+/LysMD53S and LysM–/–/LysMD53S transgenic mice, confirming that the mutant protein was secreted into the airspaces where it comprised the majority of lysozyme protein.

Immunohistochemical analyses detected lysozyme in alveolar type II cells and in alveolar macrophages of WT mice (Fig. 4C). Strong immunoreactivity was also detected in nonciliated bronchiolar epithelial cells (Clara cells) of LysM+/+/LysMD53S mice (Fig. 4C) and LysM–/–/LysMD53S mice (data not shown), suggesting that these cells were a major source of secreted lysozyme protein in the lungs of transgenic mice. Lung architecture was normal and cellular infiltration of airspaces was not detected in transgenic mice of both genotypes. Muramidase activity in BALF from LysM+/+/LysMD53S was not significantly different from that in WT mice (Fig. 4D). Muramidase activity was also decreased to the same extent (~50%) in LysM–/– mice and LysM–/–/LysMD53S transgenic mice confirming that the D53S protein was enzymatically inactive and did not alter LysP expression. Overall, expression of the D53S transgene significantly increased the level of lysozyme protein in the airspaces without increasing muramidase activity.

Bacterial killing

To assess the effect of secreted D53S protein on bacterial killing in vivo, mice from all four genotypes (WT, LysM+/+/LysMD53S, LysM–/–, and LysM–/–/LysMD53S) were intratracheally infected with P. aeruginosa and bacterial burden was assessed after 24 h (Fig. 5A). Bacterial burden was increased 3.5-fold in LysM–/– mice compared with WT mice. Expression of D53S enhanced bacterial killing 5-fold in LysM+/+/LysMD53S mice compared with WT mice and 17.5-fold compared with LysM–/– mice. Importantly, D53S protein completely restored killing of P. aeruginosa in LysM–/–/LysMD53S transgenic mice. Muramidase activity was similarly elevated in LysM–/– and LysM–/–/LysMD53S mice following infection, indicating that lysozyme P compensation did not account for restoration of pathogen killing. To determine whether D53S conferred protection against infection with another clinically important Gram-negative bacteria, mice were challenged with Klebsiella pneumoniae. Consistent with results of a previous study (11), bacterial burden in the lungs of LysM–/– mice was increased 3- to 4-fold compared with WT mice (Fig. 5B). Expression of D53S protein completely restored killing of K. pneumoniae in LysM–/– mice and enhanced bacterial killing in WT mice, similar to results for P. aeruginosa (Fig. 5A). Decreased susceptibility of LysM–/–/LysMD53S mice to infection with P. aeruginosa was reflected in histopathology at 24 h postinfection (Fig. 5C). Lung sections showed lobar pneumonia in all four groups of mice with the most severe consolidation in LysM–/– mice. P. aeruginosa was detected in all alveoli of LysM–/– mice whereas only occasional alveoli were infected in LysM–/–/LysMD53S mice. Intratracheal challenge with a larger dose of P. aeruginosa (1 x 108 CFU) resulted in death of 40% of LysM–/– mice 84 h after infection (Fig. 6). At this time point, survival was modestly (80 vs 60%) but significantly (p < 0.04) increased for infected LysM–/–/LysMD53S mice. A similar pattern was observed when mice were challenged with K. pneumoniae (data not shown). These results indicate that muramidase-deficient lysozyme protects against airway infection by clinically important Gram-negative organisms.


Figure 5
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  		FIGURE 5. Muramidase-deficient lysozyme M enhances killing of Gram-negative bacteria in transgenic mice. Five-week-old LysM+/+/LysMD53S, LysM–/–, LysM–/–/LysMD53S, and WT mice were infected with 107 CFU of P. aeruginosa (A) or 104 CFU of K. pneumoniae (B) by intratracheal instillation. Bacterial burden was assessed by quantitative culture of lung homogenates 24 h postinfection; n = 8–10 mice/group. The experiments were performed twice for each pathogen. Data are expressed as CFU per gram of lung tissue ± SEM (A). *, p < 0.02, LysM+/+/LysMD53S vs WT, **, p < 0.001, LysM–/– vs LysM–/–/LysMD53S for P. aeruginosa (B). *, p < 05, LysM+/+/LysMD53S vs WT, **, p < 0.01, LysM–/– vs LysM–/–/LysMD53S for K. pneumoniae (C). Six-week-old LysM–/– or LysM–/–/LysMD53S mice were infected with 107 CFU of P. aeruginosa by intratracheal instillation. Paraffin-embedded lung sections obtained 24 h postinfection were stained for bacteria with Brown and Brenn reagents. Arrows, bacteria.

 

Figure 6
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  		FIGURE 6. Muramidase-deficient lysozyme enhances survival following infection with Gram-negative bacteria. Twenty 5-wk-old LysM–/– or LysM–/–/LysMD53S mice were infected with 108 CFU of P. aeruginosa by intratracheal instillation. Survival was monitored for 120 h at which time surviving mice were sacrificed; p < 0.01 by log-rank test; n = 20 mice/group; *, p < 0.04.

 
The ability of D53S to enhance killing of Gram-positive bacteria was assessed after intratracheal installation of S. aureus. Bacterial burden was significantly increased in LysM–/– mice compared with WT mice at 12, 24, and 48 h after infection, although relatively few bacteria remained in the airspaces after 48 h (Fig. 7). Expression of D53S completely restored killing of S. aureus in LysM–/–/LysMD53S transgenic mice at all three time points. These results indicate that muramidase-deficient lysozyme confers protection against a clinically important Gram-positive organism in vivo.


Figure 7
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  		FIGURE 7. Muramidase-deficient lysozyme killing of Gram-positive bacteria in transgenic mice. Five-week-old WT, LysM+/+/LysMD53S, LysM–/–, and LysM–/–/LysMD53S mice were infected with 105 CFU of S. aureus by intratracheal instillation. Bacterial burden was assessed by quantitative culture of lung homogenates at 12, 24, or 48 h postinfection; n = 8–10 mice/group. Data are expressed as CFU per gram of lung tissue ± SEM. *, p < 0.04, WT and LysM–/–/LysMD53S vs LysM–/–; **, p < 0.02, WT and LysM–/–/LysMD53S vs LysM–/–; ***, p < 0.05, LysM–/–/LysMD53S and WT vs LysM–/–.

 
In a separate experiment, mice were intratracheally inoculated with S. aureus and BALF were collected at time points up to 120 h postinfection for analyses of cellular composition (Fig. 8). In WT mice, neutrophil influx peaked 12–24 h postinfection and returned to preinfection levels by 72 h. Neutrophilc influx in LysM–/– mice was dramatically elevated at 12–24 h (p < 0.01) and persisted to 120 h postinfection. Despite enhanced bacterial killing conferred by the D53S protein, inflammation in LysM–/–/LysMD53S transgenic mice was elevated and not significantly different from that in LysM–/– mice. Surprisingly, the percentage of neutrophils was also significantly elevated in BALF from LysM+/+/LysMD53S transgenic mice (p < 0.02 at 12 and 24 h). This result likely reflects the fact that the D53S protein can bind but not hydrolyze substrate and thus may act as a competitive inhibitor of endogenous lysozyme M in these mice.


Figure 8
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  		FIGURE 8. Recruitment of neutrophils to the airspace following intratracheal challenge with S. aureus. Four groups of mice, LysM+/+, LysM+/+/LysMD53S, LysM–/–, or LysM–/–/LysMD53S were infected with S. aureus. Total and differential cell counts were assessed on BALF obtained from uninfected mice (PBS) or at 12, 24, 48, 72, 96, and 120 h after infection; n = 4–5 mice/group/time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Based on the results of in vitro studies, lysozyme is widely acknowledged to be bacteriolytic for some Gram-positive bacteria with relatively little activity against Gram-negative organisms (23, 24). Resistance to the bacteriolytic effects of lysozyme has been attributed to the presence of an outer membrane in Gram-negative bacteria that limits access of the enzyme to the underlying peptidoglycan substrate. In contrast, peptidoglycan in the cell wall of Gram-positive bacteria is readily accessible to lysozyme and is consequently rapidly degraded, ultimately leading to cell lysis. These observations in vitro, coupled with the fact that lysozyme is one of the most abundant antimicrobial proteins in the lungs, have led to predictions that 1) lysozyme plays an important role in killing Gram-positive airway pathogens in vivo and 2) the peptidoglycan degrading property of lysozyme (i.e., muramidase activity) is critical for bacterial killing.

To test the importance of muramidase activity for bacterial killing in vivo, a catalytically inactive form of lysozyme was generated. Substitution of serine for aspartic acid in the active site of mouse lysozyme M resulted in complete loss of muramidase activity, as previously reported for lysozyme from other species (20, 25, 26). Muramidase-deficient recombinant lysozyme readily killed S. aureus in vitro, confirming the findings of Ibrahim et al. (20). Recombinant D53S protein also killed two Gram-negative bacteria, P. aeruginosa (Fig. 2) and K. pneumoniae (data not shown). Interestingly, the ability of recombinant lysozyme to kill Gram-positive or Gram-negative bacteria was not significantly affected by the loss of muramidase activity. Targeted expression of muramidase-deficient lysozyme in respiratory epithelial cells of LysM–/– mice significantly increased lysozyme protein concentration in the airspaces without increasing muramidase activity. Consistent with the results of in vitro assays, elevated levels of catalytically inactive lysozyme in BALF were associated with enhanced bactericidal activity against clinically important Gram-positive and Gram-negative bacteria. Intratracheal infection of LysM–/– mice with S. aureus resulted in a significant increase in bacterial burden. Importantly, expression of the D53S transgene completely restored killing of S. aureus in LysM–/– mice. Similarly, increased susceptibility of LysM–/– mice to the Gram-negative bacteria P. aeruginosa and K. pneumoniae was completely reversed by targeted expression of catalytically inactive lysozyme. Further, expression of the D53S transgene modestly but significantly enhanced survival of LysM–/– (LysM–/–/LysMD53S) mice and significantly enhanced bacterial clearance in WT (LysM+/+/LysMD53S) mice. Neutrophil influx was similarly increased in LysM–/– and LysM–/–/LysMD53S mice suggesting that the D53S protein, rather than neutrophil-derived antimicrobial peptides, accounted for the correction of bacterial killing in LysM–/– mice. Collectively, these results provide the first evidence that the muramidase-independent bactericidal activity of lysozyme plays an important role in airway host defense.

Although the concept of muramidase-independent killing of bacteria continues to be debated (27), there is increasing evidence in support of this hypothesis (20, 22, 28, 29). Recent studies identified a bactericidal domain that mapped to the C-terminal region of chicken and human lysozyme (30). Antimicrobial peptides in general are cationic at physiologic pH and associate with microbial membranes through electrostatic interactions that are often facilitated by an amphipathic helical structure. Lysozyme contains a conserved, cationic helix-loop-helix (residues 87–115 of lysozyme M and P) with potent in vitro, bactericidal activity against both Gram-positive and Gram-negative bacteria (24, 30). It is conceivable that the helix-loop-helix motif is responsible for part or all of the bactericidal activity of catalytically inactive lysozyme in transgenic mice.

The present study cannot exclude a role for muramidase activity in bacterial killing. Constitutively elevated concentration of the D53S transgene protein may partially compensate for the loss of muramidase activity by increasing the content of catalytically inactive but bactericidal lysozyme protein in the airspaces. It is also possible that muramidase activity may be required for killing of pathogens not tested in the current study. Although the role of muramidase activity in bacterial killing remains unclear, there is increasing evidence that muramidase activity may play an important role in modulating inflammation at the site of infection by rapidly degrading peptidoglycan. Peptidoglycan fragments derived from autolysis of Gram-positive cell walls induce a strong inflammatory response (31). Ganz et al. (18) demonstrated that inefficient hydrolysis of peptidoglycan in LysM–/– mice was associated with a prolonged and intense inflammatory response following s.c. injection of Micrococcus luteus, consistent with the hypothesis that muramidase activity limits inflammation associated with infection by Gram-positive organisms. This hypothesis is supported by the results of the present study which demonstrated that expression of the D53S protein completely restored killing of S. aureus without correcting the persistent, elevated inflammatory response in LysM–/– mice. The abundant muramidase activity in mouse BALF, arising from lysozyme secreted by macrophages and alveolar type II epithelial cells, as well as a nonlysozyme muramidase activity of unknown cellular origin (15), also supports an important role for peptidoglycan degradation in innate host defense of the airspaces.

Disruption of the LysM locus by insertion of GFP (17) resulted in elevated expression of lysozyme P protein in type II epithelial cells (15) and macrophages (18) but not neutrophils. Despite compensatory expression of LysP, muramidase activity in LysM–/– mice was only partially restored resulting in increased susceptibility to infection by K. pneumoniae (11) and M. luteus (18). Interestingly, lysozyme P mRNA and protein remained elevated in LysM–/–/LysMD53S transgenic mice despite abundant LysMD53S protein in type II epithelial cells and in the airspaces. Further, transcription from the LysM locus was maintained in both LysM–/– mice (15, 18) and LysM–/–/LysMD53S transgenic mice (data not shown), as indicated by detection of GFP in type II cells and macrophages. These results suggest that muramidase activity may also be an important regulator of LysP gene expression, although the pathway through which such signaling is transduced is not known.

The results of current studies in transgenic mice and previous experiments with catalytically inactive, recombinant enzyme suggest that lysozyme kills bacteria independent of its muramidase activity. This conclusion is supported by the observation that peptides derived from lysozyme and lacking muramidase activity effectively killed both Gram-positive and Gram-negative bacteria. It remains to be determined whether the muramidase-independent bactericidal property of lysozyme is generalizable to other common respiratory pathogens.


   Acknowledgments
 
We acknowledge Dr. Cindy Bachurski’s help with S1 nuclease analyses, and Ann Maher for secretarial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institutes of Health (AI/HL50797 to H.T.A.) and the Cystic Fibrosis Foundation (CFF R-457-CR02 to T.E.W.). Back

2 Address correspondence and reprint requests to Dr. Henry T. Akinbi, Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, MLC 7029, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: henry.akinbi{at}cchmc.org Back

3 Abbreviations used in this paper: SP, surfactant protein; WT, wild type; BALF, bronchoalveolar lavage fluid. Back

Received for publication October 20, 2005. Accepted for publication April 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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