Inhibition of Streptococcus pneumoniae growth by masarimycin

Abstract

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive bacterium Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide masarimycin as a bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 8 µM. The diamide inhibited detergent-induced autolysis in a concentration-dependent manner, indicating perturbation of peptidoglycan degradation as the mode-of-action. Cell based screening of masarimycin against a panel of autolysin mutants, identified a higher MIC against a ΔlytB strain lacking an endo-N-acetylglucosaminidase involved in cell division. Subsequent biochemical and phenotypic analyses suggested that the higher MIC was due to an indirect interaction with LytB. Further analysis of changes to the cell surface in masarimycin treated cells identified the overexpression of several moonlighting proteins, including elongation factor Tu which is implicated in regulating cell shape. Checkerboard assays using masarimycin in concert with additional antibiotics identified an antagonistic relationship with the cell wall targeting antibiotic fosfomycin, which further supports a cell wall mode-of-action.

Keywords: cell wall; chemical biology; meta-phenotype; mode-of-action; peptidoglycan.

Fig. 1.

Structure of PG showing the cleavage sites of several of the characterized autolysins in S. pneumoniae . Inset: structure of the antimicrobial diamide masarimycin.

Fig. 2.

Screening of the diamide masarimycin against Streptococcus pneumoniae . (a) The diamide masarimycin inhibits detergent-induced autolysis in a concentration dependent manner. Percent residual activity was calculated using autolysis in the absence of inhibitor set as 100 %. Data shown is the average of experiments performed in biological and technical triplicate. Error bars denote standard deviation. (b) Activity of masarimycin against S. pneumoniae R6 autolysin and cell wall biosynthesis mutants (13) to identify changes to masarimycin sensitivity. Assays were run in biological triplicate and yielded the same MIC values.

Fig. 3.

Chain dispersing assay with S. pneumoniae TIGR4 ΔlytB strain and purified recombinant LytB (rLytB, 2 µM). In the presence of 40 µM masarimycin dispersion of the ΔlytB chain phenotype is not inhibited. Images were taken at 1000 x magnification.

Fig. 4.

Morphological analysis of S. pneumoniae ΔlytB mutant- [15] in the presence of sub-MIC masarimycin, the β-lactam cefoxitin (DacA/PBP3 selective) or in combination. Cells were fixed in 1 % formaldehyde, stained with methylene blue, and visualized using bright field microscopy under oil immersion.