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Originally published online as doi:10.1189/jlb.0205073 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1081-1085.)
© 2005 by Society for Leukocyte Biology

Decreased clearance of Pseudomonas aeruginosa from airways of mice deficient in lysozyme M

Alexander M. Cole*,1, Dharma R. Thapa{dagger}, Victoria Gabayan{dagger}, Hsiang-I Liao{dagger}, Lide Liu{dagger} and Tomas Ganz{dagger},2

* Department of Molecular Biology and Microbiology, Biomolecular Science Center, University of Central Florida, Orlando; and
{dagger} Department of Medicine, Division of Pulmonary and Critical Care Medicine and the Will Rogers Institute Pulmonary Research Laboratory, David Geffen School of Medicine at University of California Los Angeles

1Correspondence: Department of Molecular Biology and Microbiology, Biomolecular Science Center, University of Central Florida, 4000 Central Florida Blvd., Bldg. 20, Rm. 236, Orlando, FL 32816. E-mail: acole{at}mail.ucf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Lysozyme is a ubiquitous and abundant, cationic, antimicrobial polypeptide of leukocytes and epithelia, but its biological function in host defense is largely unexplored. To ascertain the role of lysozyme during bacterial infection of murine airways, we exposed the airways of lysozyme M-deficient (lys M–/–) mice to the pulmonary pathogen Pseudomonas aeruginosa and examined the host’s response to infection. Despite partial compensation as a result of the appearance of lysozyme P in the infected airways of lys M–/– mice, these lys M–/– mice showed decreased clearance of P. aeruginosa compared with their lys M+/– or lys M+/+ littermates. Lysozyme contributes to optimal clearance of P. aeruginosa from the murine airways.

Key Words: knockout • bacteria


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
As a result of its in vitro antimicrobial activity and abundant and ubiquitous expression in airway epithelia and cells of myeloid lineage, lysozyme has long been presumed to have a role in host defense of the airways. However, only recently has direct evidence of the biological role of lysozyme been reported. In humans, lysozyme is expressed in airway (nasal) fluid at biologically active concentrations (≥500 µg/ml) and is responsible for a majority of its intrinsic antibacterial activity [1 ]. Selective removal of cationic polypeptides from nasal secretions ablated the ex vivo activity of whole nasal fluid against mucoid Pseudomonas aeruginosa, but reconstitution with physiologic concentrations of lysozyme alone could restore activity [1 ]. Akinbi and colleagues [2 ] reported that mice overexpressing exogenous rat lysozyme were more efficient than wild-type mice in killing group B streptococci and mucoid P. aeruginosa and had an increased survival rate after infection. We recently reported that mice rendered deficient in lysozyme M (lys M–/–) developed skin lesions when injected with the normally nonpathogenic and highly lysozyme-sensitive bacterium Micrococcus luteus [3 ]. Tissue injury in these mice was a result of the failure to inactivate and degrade the cell wall component peptidoglycan, which resulted in a prolonged inflammatory response.

Although humans have one lysozyme gene, mice have two different genes, which differ by only six amino acids: lysozyme M, expressed by myeloid cells and epithelia, and lysozyme P, expressed in Paneth cells of the small intestinal Crypts of Lieberkühn. Each gene product is a 14-kDa cationic enzyme, which cleaves the linkage between N-acetyl muramic acid and N-acetyl glucosamine of bacterial peptidoglycan. Although hydrolysis of peptidoglycan does not initially kill bacteria, it alters the shape and impairs the mechanical rigidity of bacteria, setting the stage for downstream events, which mechanically or chemically stress the cell. Like other cationic proteins, lysozyme has direct bactericidal activity, which is independent of its enzymatic activity [4 ]. Lysozyme may also indirectly damage bacteria by activating endogenous, autolytic enzymes within the cell wall normally responsible for remodeling during the process of cell division [5 6 7 ].

In the current study, we used lys M–/– mice to explore the role of lysozyme during P. aeruginosa infection of the murine airways. While this manuscript was in preparation, we learned of an elegant study by Markart and colleagues [8 ], which revealed that mouse lysozyme M is important for resistance to pulmonary Klebsiella pneumoniae infection. Our study complements and extends their finding that lysozyme M is a critical component of airways host defense against pathogenic microbes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Transgenic mice
lys M–/– mice were prepared as described previously [9 ], by homologous recombination of enhanced green fluorescent protein and neomycin-resistance (neo) gene into the first exon of the lysozyme M gene with the subsequent removal of the lox-flanked neo gene by passage through cre-expressing mice. The resulting transgenic mice contained contributions from C57Bl6 and 129Sv strains. To provide a homogeneous background, lys M–/– mice were backcrossed for six generations to the C57Bl6 mouse strain, and then the N6 lys M+/– mice were crossed to generate mixed litters containing wild-type (lys M+/+), lys M+/–, and lys M–/– mice. The genotype of each strain was confirmed by polymerase chain reaction and Southern analyses. Animal experiments complied with all federal and institutional (Institutional Animal Care and Use Committee) guidelines.

Bacterial culture conditions
The P. aeruginosa strain H103 was generously donated by Dr. Robert E. W. Hancock (University of British Columbia, Vancouver, Canada). P. aeruginosa H103 [10 ] was cultured 18 h at 37°C in 50 ml 3% Trypticase soy broth (TSB), snap-frozen by rapidly freezing aliquots in liquid nitrogen, and stored at –80°C for up to 1 year. Aliquots (500 µl) were thawed and subcultured 2.5 h immediately prior to use in antimicrobial assays in 50 ml 3% TSB to obtain a midlogarithmic growth phase. Subcultures were then centrifuged at 1400 g for 10 min and washed twice in 10 ml 1x Hank’s balanced salt solution (HBSS). The bacteria were collected at 1400 g for 10 min and resuspended in 1x HBSS. An optical density 625 = 0.16–0.18 approximated 2.5 x 107 bacterial colony-forming units (CFU)/ml. Exact CFU used in each experiment were determined post hoc by manually counting colonies of serial tenfold dilutions spread onto TSB agar.

Intranasal airway infection and bronchoalveolar lavage (BAL)
Mice, 6–8 weeks old, were briefly anesthetized with inhaled isoflurane, and P. aeruginosa H103 (2x107 CFU/50 µl 1xHBSS) was introduced carefully into both nares, which the mice aspirated reflexively. After 18–20 h, mice were killed with an overdose of inhaled isoflurane, and the lungs and trachea were exposed. Flexible Tygon tubing (0.060'' outer diameter, 0.020'' inner diameter, St-Gobain Performance Plastics, Akron, OH) was inserted into a 1.5- to 2.0-mm midline incision in the trachea and secured with a nylon ligature. Lungs were lavaged with 5 ml normal saline containing 5 mM EDTA. Cells were sedimented promptly at 300 g for 10 min and washed with 5 ml phosphate-buffered saline (PBS), and an aliquot of cells was resuspended in RPMI 1640 + 10% fetal calf serum. The remaining cells were kept in PBS for lysoplate analyses. Total leukocytes were counted using a hemocytometer, and a fraction of the total cells was centrifuged onto lysine-coated microscope slides using a CytoSpin3 (Shandon, Cheshire, England) at 800 rpm for 10 min and stained with DiffQuik (Dade Behring, Inc., Newark, DE) to perform a differential leukocyte count (~200 total cells per mouse, counted in randomly selected ocular fields of view). The expression of lysozyme was confirmed by immunocytochemistry, using 1:1000 rabbit antilysozyme P antisera and Fast Red chromogenic detection (Sigma Chemical Co., St. Louis, MO). Alveolar macrophages constituted >95% of total leukocytes in uninfected mice. Mice that were productively infected were determined to have >50% neutrophils in their BAL. Epithelial cells were a minor (<2%) component of the total cell count.

Western analysis of lysozyme in BAL
BAL fluids were separated into cellular and fluid (supernatant) fractions by low-speed centrifugation (300 g), and the cellular fraction was extracted separately with 5% acetic acid. The acid extract was lyophilized and resuspended in 0.01% acetic acid. The extracts and the fluid fractions were then absorbed with MacroPrep CM beads (Bio-Rad, Hercules, CA) at pH 6, and the bound material was eluted with 5% acetic acid and centrifuged through a 3-kDa molecular weight cutoff filter (Centricon, Millipore, Bedford, MA) to remove excess salts. The retentate volume was adjusted to 100 µL in 0.01% acetic acid, separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [11 ], and immunoblotted to Immobilon PSQ-polyvinylidene difluoride membranes (Millipore) for 1 h in 0.7% acetic acid/10% methanol, as described previously [12 ]. The membranes were then incubated with 1:1000 dilution of rabbit polyclonal anti-mouse lysozyme M or anti-mouse lysozyme P overnight at room temperature. After several washings in 1x Blotto Tris-buffered saline plus 1% dried milk and 0.1% bovine serum albumin, membranes were incubated with a 1:20,000 dilution of peroxidase-conjugated polyclonal goat anti-rabbit secondary antibody (Pierce, Rockford, IL) for 1 h at room temperature. The membranes were washed and then developed using Supersignal West Pico chemiluminescent substrate (Pierce). Recombinant lysozyme M and P protein standards were produced in a baculovirus expression system as described previously [3 , 13 ] and were used as standards for Western analyses. Rabbit anti-mouse lysozyme M and P polyclonal antisera were produced as described previously [3 , 13 ].

Lysoplate assay for muramidase activity of lysozyme M and lysozyme P
Briefly, 0.5 mg/ml lyophilized M. luteus was suspended in 66 mM sodium phosphate buffer (Na2HPO4/NaH2PO4, pH 7.4), combined with 10 ml 1% low electroendoosmotic (EEO) agarose in 66 mM sodium phosphate, and poured in a level, 9 x 9 cm square petri dish [14 ]. Sixteen or 25 evenly spaced 3.2 mm wells were punched in the solidified agar, and 5 µl sample was introduced into each well (size of well is 7–8 µl). Samples (in 1xPBS) included lysozyme M standard, lysozyme P standard, cell-free BAL fluid, or total cells recovered from BAL. To recover lysozyme, cells were washed in PBS, extracted with 5% acetic acid, and sonicated for 10 s x two bursts. Clarified supernatant was lyophilized and resuspended in 50 µl 0.01% acetic acid. Lysozyme enzymatic activity was determined by measuring the diameters of zones of clearance relative to recombinant standards of lysozyme M or lysozyme P. To calculate the concentration of lysozyme per cell, total lysozyme concentration was divided by the number of cells enumerated with a hemacytometer. Recombinant lysozyme M and P standards were generated as described previously by our group [3 ].

Radial diffusion assay to determine antipseudomonal activity of lysozyme M and lysozyme P
Radial diffusion assays (RDA) were performed as described previously [15 , 16 ]. Briefly, the underlay consisted of 1% low EEO agarose and 1:100 dilution of TSB in 10 mM sodium phosphate, pH 7.4. The overlay consisted of 6% TSB and 1% agarose in distilled H2O for all assays. P. aeruginosa strain H103 (4x106) was mixed with 10 ml underlay gel solutions, kept molten at 48°C, and poured into 100 cm2 square petri dishes. A series of 3.2 mm diameter wells was punched after the agarose solidified, and 5 µl lysozyme sample was added into designated wells. Plates were incubated at 37°C for 3 h to allow for protein diffusion. The microbe-laden underlay was then covered with 10 ml molten overlay, and the plates were allowed to harden. Antimicrobial activity was identified as a clear zone around the well, devoid of microbial growth after 18 h incubation at 37°C. The x-intercept of the relationship between the zone diameter versus logarithm to the base 10 (log10) peptide concentration was determined by least mean squares regression and equated to the minimal inhibitory concentration (MIC).

Statistics
Comparisons between groups were analyzed by t-test or a one-way ANOVA with Tukey test (SigmaStat, SPSS Inc., Chicago, IL). For data that were not distributed normally, a log10transformation or nonparametric analysis (Mann-Whitney test) was performed. Error bars represent standard deviation.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
lys M–/– mice show decreased clearance of P. aeruginosa infection from their airways
As described previously [3 ], conventionally housed lys M–/– mice appeared healthy. To determine if lys M–/– mice were more susceptible to airways infection, lys M–/– mice and their wild-type and lys M+/– littermates were subjected to airways infection by P. aeruginosa strain H103 (2x107 CFU in 50 µl 1xHBSS). After 18–20 h, the lungs were lavaged, and P. aeruginosa CFU were quantified in triplicate and averaged for each mouse (Fig. 1 ). The number of bacteria was 66-fold greater in lys M–/– mice (2.1x105 CFU/ml; n=10) than wild-type mice (3.2x103 CFU/ml; n=11; P=0.007; one-way ANOVA with Tukey test). There was also a statistically nonsignificant trend toward an increase of CFU in the lys M–/– mice as compared with the heterozygote mice (P=0.052). The quantity of recovered CFU did not differ between lys M+/– mice (n=8) and wild-type mice (n=11; P=0.822). These data indicate that lys M–/– mice have impaired clearance of P. aeruginosa from the airways.



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  		Figure 1. lys M–/– mice show impaired clearance of P. aeruginosa airways infection. The airways of lys M–/– (–/–), lys M+/– (+/–), and wild-type mice (+/+) were infected with 2 x 107 CFU P. aeruginosa. CFU were recovered from BAL 18–20 h postinfection and quantitated. Each point represents a single mouse, and the dashed lines indicate mean ± SD. BAL fluid from lys M–/– mice had a significantly higher concentration of viable P. aeruginosa than wild-type control.

 
The inflammatory response is not dysregulated in lys M–/– mice
We next assayed the total number of cells and the percentage of neutrophils in BAL fluid (Fig. 2 ) to determine whether the inflammatory response of lys M–/– mice was altered and whether such a change could account for the observed impairment of antimicrobial activity in vivo. For total cell count as well as percent neutrophils, there was no significant difference amongst lys M–/– mice (n=10), hemizygous mice (n=8), and wild-type, control mice (n=11), and thus, the recruitment of leukocytes in general and neutrophils in particular was not impaired in lys M–/– mice. The impaired clearance of P. aeruginosa from the airways was thus a result of the diminished antimicrobial potency of the lys M–/– leukocytes and airways epithelia.



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  		Figure 2. Total cell count and number of neutrophils are similar in lys M–/– and sufficient mice. Total cell count per ml of BAL fluid (A) and percent neutrophils (B) was determined for lys M–/–, lys M+/–, and wild-type mice at 18–20 h. Each point represents a single mouse, and the dashed lines indicate mean ± SD. Note that the number of total cells per ml in BAL fluid as well as the percentage of neutrophils were not significantly different among the three groups of mice.

 
Lysozyme P expression in the airways of lys M–/– mice is not sufficient to compensate for the loss of lysozyme M activity
Although we previously showed that in uninfected lys M–/– mice [3 ], alveolar macrophages partially compensate by producing lysozyme P, the expression of lysozyme M or P during airways infection has not been explored. Total cells (>98% neutrophils) were obtained from BAL fluid of P. aeruginosa-infected lys M–/–, lys M+/–, and wild-type mice and subjected to immunocytochemistry with rabbit anti-mouse lysozyme P polyclonal antisera. Antilysozyme polyclonal antibodies react with lysozyme M and P, regardless of which lysozyme immunogen (M or P) was used to produce the antibody (data not shown). Neutrophils from infected lys M–/– mice do not express lysozyme (Fig. 3A ), but neutrophils from lys M+/– mice (Fig. 3B) and wild-type mice (Fig. 3C) express appreciable amounts of lysozyme. As expected, neutrophils from wild-type mice stained with preimmune antisera were not immunoreactive (Fig. 3D) .



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  		Figure 3. Lysozyme P in the airways of lys M–/– mice is not derived from neutrophils. Cells obtained from BAL of a lys M–/– mouse (A), a lys M+/– mouse (B), and a wild-type mouse (C) infected with P. aeruginosa were stained with antilysozyme antibody and counterstained with hematoxylin. Reactive cells stained red. (D) Cells from the same wild-type mouse as in C stained with preimmune antisera and counterstained with hematoxylin. (E) Tricine SDS-PAGE Western analysis of the fluid (upper panel) and cellular (lower panel) portions of BAL fluid from lys M–/– (–/–), lys M+/– (+/–), and wild-type (+/+) mice. Lys M and Lys P, 100 ng and 200 ng each of lysozyme M and lysozyme P standards, respectively. Results are representative of three separate experiments with different animals for each replicate.

 
We next sought to determine whether cells from each mouse genotype expressed lysozyme M, lysozyme P, or both. The cellular and fluid portions of BAL were separated by centrifugation (300 g), and ~106 cells and 3, 5, and 10 µl supernatant from wild-type, lys M+/–, and lys M–/–, respectively, were subjected to Tricine SDS-PAGE antilysozyme Western analysis (Fig. 3E) . Lysozyme M and P can be distinguished by a slight difference in electrophoretic mobility (Lys M and Lys P standards in Fig. 3E ), similar to the shift seen when the two proteins were electrophoresed in acid urea-PAGE [3 ]. As expected, lysozyme M was found in the fluid and cellular components of BAL from wild-type mice and was absent in both components of BAL from lys M–/– mice. Instead, lys M–/– mice expressed lysozyme P in the fluid fraction but not in the cellular fraction, indicating that neutrophils are not the source of lysozyme P during airways infection. It is interesting that lysozyme M was also the solitary lysozyme component in the humoral and cellular fractions of lys M+/– mice.

Lysozyme concentration was quantified separately for the cellular and fluid components of BAL by lysoplate analysis (Fig. 4 ). Based on results from Figure 3 , lysozyme M standards were used to quantify samples from wild-type (n=10) and hemizygous (n=8) mice, and lysozyme P standards were used to quantify samples from lys M–/– mice (n=11). For the cellular components of BAL, the lysozyme content per neutrophil was significantly reduced in lys M–/– mice as compared with lys M+/– mice (P=0.0025) and wild-type mice (P=0.00014). Likewise, lysozyme concentration of the fluid (supernatant) fraction of BAL from lys M–/– mice was significantly lower than comparable fractions from lys M+/– (P=0.018) or wild-type (P<0.0001) mice. Concentration of lysozyme was not statistically different between lys M+/– and wild-type mice (P=0.12–0.24). Although measurements of lysozyme concentration in BAL were associated with muramidase activity, they did not account for nonlysozyme muramidase activity, which could possibly be present in the fluid. Nevertheless, as the lysozyme measurements were clearly different in the three types of mice, background muramidase activity was likely not a significant factor. Collectively, the lower levels of total lysozyme in lys M–/– mice may account for the reduced in vivo antimicrobial activity against P. aeruginosa H103 (Fig. 1) .



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  		Figure 4. Concentration of lysozyme in BAL is diminished in lys M–/– mice. The concentration of lysozyme in the cellular (A) and fluid (B) component of BAL was determined by lysoplate analysis for lys M–/–, lys M+/–, and lys M+/+ mice. To calculate the concentration of lysozyme per cell, total lysozyme concentration was divided by the number of cells enumerated with a hemocytometer. Each point represents a single mouse, and the dashed lines indicate mean ± SD.

 
Another contributing factor to the reduced antimicrobial activity in the airways of lys M–/– mice is that the antipseudomonal activity of lysozyme P is significantly lower than the activity of lysozyme M (Fig. 5 ). Both forms of lysozyme were subjected to a RDA against P. aeruginosa H103, and the MIC of lysozyme P was determined in five separate experiments to be significantly higher than lysozyme M (P=0.034). The combination of lower levels of lysozyme P expression in lys M–/– mice, coupled with the lower activity of lysozyme P, indicates that the deficiency of lysozyme M in lys M–/– mice can only be partially alleviated by the expression of lysozyme P.



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  		Figure 5. Comparison of antipseudomonal activity of lysozyme P and lysozyme M. Purified recombinant lysozyme P and M were subjected to RDAs against P. aeruginosa H103, and the MIC for each protein was calculated. Error bars indicate SD.

 
A final factor to be considered is that the cellular distribution of lysozyme activity is radically altered in lys M–/– mice. In these mice, lysozyme P is expressed by alveolar macrophages [3 , 17 ] and type II alveolar cells [17 ] but not in neutrophils, which act as primary host defense cells during a massive acute infection. The compensatory expression of lysozyme P may be sufficient to protect the mice against infection with less virulent organisms or small numbers of pathogens and may explain why lys M–/– mice are not appreciably susceptible to spontaneous infection. However, the absence of lysozyme from neutrophils may disproportionately affect the host response to a massive infection with a virulent organism, a situation in which fully functioning neutrophils are essential.

Our study and that of Markart et al. [8 ] contradict the commonly held notion that lysozyme is only important in host defense against nonpathogenic, highly lysozyme-sensitive, gram-positive bacteria. The high concentrations of this antimicrobial enzyme in the airways and in phagocytes [1 , 10 ] and its additive and synergistic interactions with other antimicrobial components in leukocytes and epithelia [18 ] enhance its importance even against microbes that are not highly sensitive to its effects in vitro.


   ACKNOWLEDGEMENTS
 
This work was supported by Grants R01 HL70876 (to A. M. C.) and R01 AI48167 and P50 HL67665 (to T. G.) from the National Institutes of Health, a research training grant from the American Lung Association (to A. M. C.), and grants from the Cystic Fibrosis Foundation and Cystic Fibrosis Research, Inc. (to T. G. and A. M. C.). The authors thank Dr. Thomas Graf for generously providing the initial lys M–/– mice for our breeding program.


   FOOTNOTES
 
2 Correspondence: Department of Medicine, Division of Pulmonary and Critical Care Medicine, 10833 Le Conte Ave., CHS 37-055, Los Angeles, CA 90095. E-mail: tganz{at}mednet.ucla.edu Back

Received February 4, 2005; revised July 14, 2005; accepted July 15, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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  7. Ginsburg, I. (2004) Bactericidal cationic peptides can also function as bacteriolysis-inducing agents mimicking ß-lactam antibiotics?; it is enigmatic why this concept is consistently disregarded Med. Hypotheses 62,367-374[CrossRef][Medline]
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  9. Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. (2000) Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages Blood 96,719-726[Abstract/Free Full Text]
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  17. Markart, P., Faust, N., Graf, T., Na, C. L., Weaver, T. E., Akinbi, H. T. (2004) Comparison of the microbicidal and muramidase activities of mouse lysozyme M and P Biochem. J. 380,385-392[CrossRef][Medline]
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