Investigation of the mechanism of action of a pair of novel antibiotics in the gram-negative bacterium Escherichia coli

<p dir="ltr">A lack of novel antibiotic development has been linked to the burgeoning antimicrobial resistance crisis due to emerging resistance against existing classes of antibiotics. To this end, the GKL compounds were developed as a novel class of antibiotics designed to target transcription in bacteria. A pair of these antimicrobials, while effective at killing gram-positive bacteria, have been found to lack anti-transcription activity, and thus have alternative mode(s) of antimicrobial action. This study was carried out to investigate the mechanisms of action of these compounds in the Gram-negative bacterium Escherichia coli. The investigation was carried out using the functional genomics techniques RNA Sequencing (RNA-Seq) and transposon-directed insertion site sequencing (TraDIS) to investigate the global cellular responses to these compounds.</p><p dir="ltr">The RNA-Seq experiments, in concert with a range of bioinformatics analyses, highlighted novel transcriptomic responses of E. coli to these compounds, as well as a range of potential mechanisms of transport of these compounds into the cytosol of E. coli. The function of candidate genes identified through RNA-Seq were investigated through phenotypic analysis of deletion mutants, and a pair of inner membrane purine transporters, GhxP and AdeP, were identified as being linked to the transport of GKL277 into the cytosol. While proteoliposome transport assays were unable to determine the transport of GKL277 by GhxP, the information gained from these phenotypic analyses significantly increases our understanding of these compounds, as well as indicates the high likelihood of the cytosolic locality of the GKL277 target.</p><p dir="ltr">These analyses identified a range of pathways impacted by exposure to the GKL compounds, informing future work. In addition, these analyses highlight potential future use cases of these compounds, such as the treatment of E. coli urinary tract infections.</p><p dir="ltr">The construction of a Tn5 transposon mutagenesis library in this study demonstrated the use of the pDS1028 conjugative transposon delivery system in E. coli BW25113-Imp, which has not been previously demonstrated in this strain. While the library generated in this study was of substantial mutant density, the transposon insertion bias demonstrated in this library presented a number of obstacles for candidate gene analysis. However, subsequent analysis of the TraDIS insertion site density identified genes that may impact the fitness of E. coli BW25113-Imp which will inform future investigation into this unique strain of E. coli.</p><p dir="ltr">Overall, this study resulted in substantial increases into the body of knowledge about the physiological responses of bacteria to the GKL compounds and identified the likely pathway of uptake of these compounds into bacteria via a pair of transporters previously unassociated with antibiotic transport. Lastly, the creation of a TraDIS library in E. coli BW25113-Imp resulted in unique findings that will inform future TraDIS library construction in this strain, as well as genes potentially involved in the fitness of the E. coli BW25113-Imp strain.</p>