Figure 3

Depletion of muramidase activity and antimicrobial activity to S. pneumoniae 6B in the E-tube homogenate of lysozyme M ^-/- mice. The lysoplate radial assay of the E-tube homogenate shows depletion of muramidase activity in lysozyme M^-/- mice (A). The lysoplate was containing lyophilized Micrococcus lysodeiktikus, a substrate of muramidase. The muramidase activity of the tissue homogenate (1 μg) is expressed relative to that of a chicken egg white lysozyme as a standard (B). The gel-overlay assays with the lysoplate demonstrate that the E-tube homogemate of lysozyme M^-/- mice lacks muramidase activity (arrow) at the corresponding level of lysozyme (C). The liquid broth assay shows antimicrobial activity of human and chicken lysozyme against S. pneumoniae in a dose-dependent manner (D). Antimicrobial activity of the E-tube homogenate decreases in lysozyme M^-/- mice, compared to wild type mice (E). 2.5 × 10⁴ CFU/mL of S. pneumoniae 6B were incubated with or without the E-tube homogenate (100 μg/mL) for 2 hours at 37°C. Con: the vehicle control, incubated without the E-tube homogenate. Values are given as mean ± standard deviation. All experiments were triplicated. *: p < 0.05, **: p < 0.01. The gel-overlay assays with the bacterial lawn of S. pneumoniae 6B demonstrate that antimicrobial activity of the E-tube homogenate (arrow) is decreased in the lysozyme M^-/- mice compared to the wild type mice (F).