Perturbation of ribosomal subunits dynamics by inhibitors of tRNA translocation

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA translocation), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, EF-G-induced transition to the rotated/swiveled state of the SSU is hardly affected. The major effect of the antibiotics is observed at the stage when the SSU body and the head domain move backward. Vio, Spc and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

Mechanistic origin of drug interactions between translation-inhibiting antibiotics

SummaryAntibiotics that interfere with translation, when combined, interact in diverse and difficult-to-predict ways. Here, we demonstrate that these interactions can be accounted for by “translation bottlenecks”: points in the translation cycle where antibiotics block ribosomal progression. To elucidate the underlying mechanisms of drug interactions between translation inhibitors, we generated translation bottlenecks genetically using inducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimicked antibiotic action and their interactions, supporting that the interplay of different translation bottlenecks causes these interactions. We further showed that the kinetics of drug uptake and binding together with growth laws allows direct prediction of a large fraction of observed interactions, yet fails for suppression. Simultaneously varying two translation bottlenecks in the same cell revealed how the dense traffic of ribosomes and competition for translation factors results in previously unexplained suppression. This result highlights the importance of “continuous epistasis” in bacterial physiology.

5-Benzylidene-4-Oxazolidinones are Synergistic with Antibiotics for the Treatment of Staphylococcus Aureus Biofilms

<div>The failure of frontline antibiotics in the clinic is one of the most serious threats to human health and requires a multitude of novel therapeutics and innovative treatment approaches to curtail the growing crisis. In addition to traditional resistance mechanisms resulting in the lack of efficacy of many antibiotics, most chronic and recurring infections are further made tolerant to antibiotic action by the presence of biofilms. Herein, we report an expanded set of 5-benzylidene-4-oxazolidinones that are able to inhibit the formation of Staphylococcus aureus biofilms, disperse preformed biofilms and in combination with common antibiotics are able to significantly reduce the bacterial load in a robust collagen-matrix model of biofilm infection.</div>