Towards an Understanding of the Difference Between Chemical and Genetic Inactivation of Bacterial Autolysins

Many of our most successful antibiotics, like penicillin, are inhibitors of bacterial cell wall synthesis, yet many bacteria have developed antibiotic resistance to them. In an updated report released by the Centers for Disease Control (CDC) in November of 2019, it was estimated that 2.8 million infections and 35,000 deaths occur in the United States alone because of antibiotic resistant bacteria. It is crucial that novel antibiotics are developed in order to fight this global crisis. Diamide antibiotics have shown promise as an antimicrobial molecular scaffold targeting the degradation of the bacterial cell wall. The diamide masarimycin works as a small molecule autolysin inhibitor that exhibits antimicrobial activity against Bacillus subtilis and Streptococcus pneumoniae in the low micromolar range. The purpose of this study is to investigate how "non-essential" enzymes, such as autolysins, can halt bacterial cell growth when enzymatic activity is inhibited. This study analyzed how these newly discovered diamide antibiotics alter the expression of enzymes involved in cell wall metabolism and how this compares to genetic inactivation of these same enzymes. In B. subtilis masarimycin inhibits the autolysin LytG, an exo-acting N-acetylglucosaminidase (GlcNAcase) that is the major active GlcNAcase during vegetative growth. It was hypothesized that chemical inactivation of LytG by masarimycin results in a unique autolysin expression profile compared to genetic inactivation of the lytG gene. Initial experiments have focused on quantifying the changes in expression levels of the major autolysins (LytC, LytD, LytG, LytF) in B. subtilis 3610 and autolytic mutants by qPCR to look for changes in expression of these enzymes after chemical or genetic inactivation. Results indicate at early exponential phase (OD600nm 0.2) B. subtilis 3610 appears to compensate for masarimycin inhibition of LytG by expressing more LytD, which could explain the heightened masarimycin sensitivity that has been observed in the lytD mutant.