The abundance of lysozyme on mucosal surfaces suggests that successful colonizers must be able to evade its antimicrobial effects. Lysozyme has a muramidase activity that hydrolyzes bacterial peptidoglycan and a non-muramidase activity attributable to its function as a cationic antimicrobial peptide. Two enzymes (PgdA, a N-acetylglucosamine deacetylase, and Adr, an O-acetyl transferase) that modify different sites on the peptidoglycan of Streptococcus pneumoniae have been implicated in its resistance to lysozyme in vitro. Here we show that the antimicrobial effect of human lysozyme is due to its muramidase activity and that both peptidoglycan modifications are required for full resistance by pneumococci. To examine the contribution of lysozyme and peptidoglycan modifications during colonization of the upper respiratory tract, competition experiments were performed with wild-type and pgdAadr mutant pneumococci in lysozyme M-sufficient (LysM+/+) and -deficient (LysM？/？) mice. The wild-type strain out-competed the double mutant in LysM+/+, but not LysM？/？ mice, indicating the importance of resistance to the muramidase activity of lysozyme during mucosal colonization. In contrast, strains containing single mutations in either pgdA or adr prevailed over the wild-type strain in both LysM+/+ and LysM？/？ mice. Our findings demonstrate that individual peptidoglycan modifications diminish fitness during colonization. The competitive advantage of wild-type pneumococci in LysM+/+ but not LysM？/？ mice suggests that the combination of peptidoglycan modifications reduces overall fitness, but that this is outweighed by the benefits of resistance to the peptidoglycan degrading activity of lysozyme.
References
[1]
Cole A, Liao H, Stuchlik O, Tilan J, Pohl J, et al. (2002) Cationic polypeptides are required for antibacterial activity of human airway fluid. J Immunol 169: 6985–6991.
[2]
Welsh I, Spitznagel J (1971) Distribution of lysosomal enzymes, cationic proteins, and bactericidal substances in subcellular fractions of human polymorphonuclear leukocytes. Infect Immun 4: 97–102.
[3]
Markart P, Korfhagen T, Weaver T, Akinbi H (2004) Mouse lysozyme M is important in pulmonary host defense against Klebsiella pneumoniae infection. Am J Respir Crit Care Med 169: 454–458.
[4]
Cramer E, Breton-Gorius J (1987) Ultrastructural localization of lysozyme in human neutrophils by immunogold. J Leukoc Biol 41: 242–247.
[5]
Ibrahim H, Matsuzaki T, Aoki T (2001) Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function. FEBS Lett 506: 27–32.
[6]
Nash J, Ballard T, Weaver T, Akinbi H (2006) The peptidoglycan-degrading property of lysozyme is not required for bactercidal activity in vivo. J Immunol 177: 519–526.
[7]
Ibrahim H, Thomas U, Pellegrini A (2001) A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. J Biol Chem 276: 43767–43774.
[8]
Akinbi H, Epaud R, Bhatt H, Weaver T (2000) Bacterial killing is enhanced by expression of lysozyme in the lungs of transgenic mice. J Immunol 165: 5760–5766.
[9]
Cole A, Thapa D, Gabayan V, Liao H, Liu L, et al. (2005) Decreased clearance of Pseudomonas aeruginosa from airways of mice deficient in lysozyme M. J Leukoc Biol 78: 1081–1085.
[10]
Vollmer W, Tomasz A (2000) The pgdA gene encodes for a peptidoglycan N-acetylglucosamine deacetylase in Streptococcus pneumoniae. J Biol Chem 275: 20496–20501.
[11]
Raymond J, Mahapatra S, Crick D, Pavelka M Jr (2005) Identification of the namH gene, encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan. J Biol Chem 280: 326–333.
[12]
Psylinakis E, Boneca I, Mavromatis K, Deli A, Hayhurst E, et al. (2005) Peptidoglycan N-acetylglucosamine deacetylases from Bacillus cereus, highly conserved proteins in Bacillus anthracis. J Biol Chem 280: 30856–30863.
[13]
Herbert S, Bera A, Nerz C, Kraus D, Peschel A, et al. (2007) Molecular basis of resistance to muramidase and cationic antimicrobial peptide activity of lysozyme in staphylococci. PLoS Pathog 3: e102. doi:10.1371/journal.ppat.0030102.
[14]
Hébert L, Courtin P, Torelli R, Sanguinetti M, Chapot-Chartier M, et al. (2007) Enterococcus faecalis constitutes an unusual bacterial model in lysozyme resistance. Infect Immun 75: 5390–5398.
[15]
Fukushima T, Kitajima T, Sekiguchi J (2005) A polysacchride deacetylase homologue, PdaA in Bacillus subtilis acts as an N-acetylmuramic acid deacetylase in vitro. J Bacteriol 187: 1287–1292.
[16]
Boneca I, Dussurget O, Cabanes D, Nahori M, Sousa S, et al. (2007) A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system. Proc Natl Acad Sci 104: 997–1002.
[17]
Blundell J, Smith G, Perkins H (1980) The peptidoglycan of Neisseria gonorrhoeae: O-acetyl groups and lysozyme sensitivity. FEMS Microbiol Lett 9: 259–261.
[18]
Bera A, Herbert S, Jakob A, Vollmer W, G？tz F (2005) Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol Microbiol 55: 778–787.
[19]
Bera A, Biswas R, Herbert S, Kulauzovic E, Weidenmaier C, et al. (2007) Influence of wall teichoic acid on lysozyme resistance in Staphylococcus aureus. J Bacteriol 189: 280–283.
[20]
S？rensen U, Henrichsen J, Chen H, Szu S (1990) Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods. Microb Pathog 8: 325–334.
[21]
Crisóstomo M, Vollmer W, Kharat A, Inhülsen S, Gehre F, et al. (2006) Attenuation of penicillin resistance in a peptidoglycan O-acetyl transferase mutant of Streptococcus pneumoniae. Mol Microbiol 61: 1497–1509.
[22]
Vollmer W, Tomasz A (2002) Peptidoglycan N-acetylglucosamine deacetylase, a putative virulence factor in Streptococcus pneumoniae. Infect Immun 70: 7176–7178.
[23]
Cross M, Mangelsdorf I, Wedel A, Renkawitz R (1988) Mouse lysozyme M gene: Isolation, characterization, and expression studies. Proc Natl Acad Sci 85: 6232–6236.
[24]
Kim J, Weiser J (1998) Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae. J Infect Dis 177: 368–377.
[25]
Magee A, Yother J (2001) Requirement for capsule in colonization by Streptococcus pneumoniae. Infect Immun 69: 3755–3761.
[26]
Nelson A, Roche A, Gould J, Chim K, Ratner A, et al. (2007) Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance. Infect Immun 75: 83–90.
[27]
Kovács M, Halfmann A, Fedtke I, Heintz M, Peschel A, et al. (2006) A function dlt operon, encoding proteins required for incorporation of D-alanine in teichoic acids in Gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae. J Bacteriol 188: 5797–5805.
[28]
Redfield C, Dobson C (1990) 1H NMR studies of human lysozyme: spectral assignment and comparison with hen lysozyme. Biochemistry 29: 7201–7214.
[29]
Peters C, Kruse U, Pollwein R, Grzeschik K, Sippel A (1989) The human lysozyme gene. Sequence organization and chromosomal localization. Eur J Biochem 182: 507–516.
[30]
Jung A, Sippel A, Grez M, Schütz G (1980) Exons encode functional and structural units of chicken lysozyme. Proc Natl Acad Sci 77: 5759–5763.
[31]
Vollmer W (2008) Structural variation in the glycan strands of bacterial peptidoglycan. FEMS Microbiol Rev 32: 287–306.
[32]
Zipperle G Jr, Ezzell J Jr, Doyle R (1984) Glucosamine substitution and muramidase susceptibility in Bacillus anthracis. Can J Microbiol 30: 553–559.
[33]
Weadge J, Clarke A (2006) Identification and characterization of O-acetylpeptidoglycan esterase: a novel enzyme discovered in Neisseria gonorrhoeae. Biochemistry 45: 839–851.
[34]
Logardt I, Neujahr H (1975) Lysis of modified walls from Lactobacillus fermentum. J Bacteriol 124: 73–77.
[35]
Araki Y, Fukuoka S, Oba S, Ito E (1971) Enzymatic deacetylation of N-acetylglucosamine residues in peptidoglycan from Bacillus cereus cell walls. Biochem Biophys Res Commun 45: 751–758.
[36]
Inohara N, Ogura Y, Fontalba A, Gutierrez O, Pons F, et al. (2003) Host recognition of bacterial muramyl dipeptide mediated through Nod2: Implications for crohn's disease. J Biol Chem 278: 5509–5512.
[37]
Girardin S, Boneca I, Viala J, Chamaillard M, Labigne A, et al. (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem 278: 8869–8872.
[38]
Girardin S, Boneca I, Carneiro L, Antignac A, Jéhanno M, et al. (2003) Nod1 detects a unique muropeptide from Gram-negative bacterial peptidoglycan. Science 300: 1584–1587.
[39]
Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S, et al. (2003) An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol 4: 702–707.
[40]
Tettelin H, Nelson K, Paulsen I, Eisen J, Read T, et al. (2001) Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293: 498–506.
[41]
Sung C, Li H, Claverys J, Morrison D (2001) An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67: 5190–5196.
[42]
LeDeaux J, Grossman A (1995) Isolation and characterization of kinC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinase KinA and KinB in Bacillus subtilis. J Bacteriol 177: 166–175.
[43]
Pericone C, Bae D, Shchepetov M, McCool T, Weiser J (2002) Short-sequence tandem and nontandem DNA repeats and endogenous hydrogen peroxide production contribute to genetic instability of Streptococcus pneumoniae. J Bacteriol 184: 4392–4399.
[44]
Faust N, Varas F, Kelly L, 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.
[45]
Hestdal K, Ruscetti F, Ihle J, Jacobsen S, Dubois C, et al. (1991) Characterization and regulation of RB6-8C5 antigen expression on murine bone marrow cells. J Immunol 147: 22–28.
[46]
Bryder D, Sasaki Y, Borge O, Jacobsen S (2004) Deceptive multilineage reconstitution analysis of mice transplanted with hemopoietic stem cells, and implications for assessment of stem cell numbers and lineage potentials. J Immunol 172: 1548–1552.
[47]
Lysenko E, Ratner A, Nelson A, Weiser J (2005) The role of innate immune responses in the outcome of interspecies competition for colonization of mucosal surfaces. PLoS Pathog 1: e1. doi:10.1371/journal.ppat.0010001.
