Genetic evidence that antibacterial activity of lysozyme is independent of its catalytic function

Abstract

A catalytically inactive mutant of hen egg white lysozyme was constructed by site-directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram-positive bacteria. The catalytic residue aspartic acid at position 52 of lysozyme was substituted with serine (D52S-Lz) and the mutant cDNA was inserted into a yeast expression vector, pYES-2. Western blot analysis indicated that the mutation did not affect secretion of the D52S-Lz lysozyme into the medium of the expressing Saccharomyces cerevisiae, INVSC1. In addition, circular dichroism and fluorescence spectral analysis revealed no change in the structure of D52S-Lz compared to that of wild-type (Wt-Lz) lysozyme. The mutation (D52S) abolished the catalytic activity of lysozyme. Antimicrobial tests against Staphylococcus aureus and Bacillus subtilis revealed that the catalytically inactive D52S-Lz was as bactericidal as the Wt-Lz lysozyme. Heat treatment leading to enzyme inactivation had no effect on the bactericidal activity of either wild-type or the mutant D52S-Lz lysozyme. The binding affinity of D52S-Lz to the isolated peptidoglycan of S. aureus was unaffected. Our results provide the first demonstration of direct genetic evidence that the antimicrobial activity of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors. ß

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

“…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).…”

“…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 M D53S construct was generated and expressed in type II cells of WT and LysM Ϫ/Ϫ mice.…”

“…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).…”

pH modulates the activity and synergism of the airway surface liquid antimicrobials β-defensin-3 and LL-37

“…Synergistic effects may occur when one peptide in a conformation parallel to the bacterial membrane facilitates insertion of other peptides (63). We speculate that such an effect might account for synergistic activity between hBD-3 and LL-37 as well as synergism for the combination of LL-37 and lysozyme; in addition to its enzymatic activity, lysozyme has nonenzymatic antibacterial activity dependent on its cationic and hydrophobic domains that disrupt bacterial plasma membranes (64)(65)(66). A possibility that we cannot exclude is that variations in pH might render S. aureus and P. aeruginosa more or less susceptible to antimicrobial factors.…”

Ixodes scapularistick saliva proteins sequentially secreted every 24h during blood feeding

“…On the other hand, proteins identified in BD and SD saliva such as fibrinogen and protease inhibitors are likely associated with events toward the end of tick feeding. For instance, functionally annotated antimicrobial peptides: antimicrobial protein CAP18 [161,162] identified in all samples except 120 h saliva, neutrophil gelatinase-associated lipocalin [163,164], neutrophil granule protein [165][166][167], protein S100-A12 [168], neutrophil antibiotic [165], and lysozyme C [169,170] that were identified in 48 h and other stage saliva (S2 Table) could aid the tick to clear microbes from the tick feeding site. It is interesting to note that we identified both tick-and rabbit-derived antimicrobial peptides at the same time points.…”