Discovering novel antimicrobial agents that target sRNA regulated antibiotic resistance mechanisms

The rise of antibiotic resistant pathogenic bacteria represents a major threat to human health that demands the development of new strategies to combat resistance mechanisms. A significant challenge is overcoming bacteria?s ability to limit the efficacy of antibiotics by reducing their intracellular concentration by increasing efflux out of the cell and decreasing entry through membrane proteins. One method bacteria use to induce these intrinsic resistance processes is through post-transcriptional regulation of genes by small RNAs (sRNAs). For example, the sRNA MicF represses the translation of outer membrane protein F (OmpF), a porin involved in antibiotic uptake. Transcription of MicF is known to increase due to environmental factors such as oxidative stress and the presence of antibiotics. Here we propose an innovative antimicrobial treatment strategy whereby we introduce a biomolecule designed to sequester MicF, thus increasing the efficacy of existing antibiotics by increasing antibiotic uptake through the porin OmpF. To achieve this goal, we will first characterize changes in the transcription of MicF during Escherichia coli?s response to representatives from the major classes of antibiotics (Aim 1). Next, we will pursue two parallel strategies to design biomolecules that will bind and sequester MicF in the cell. The first will be to design antisense molecules that bind directly to MicF (Aim 2). In the second strategy, we will randomize amino acids of known RNA binding peptides to change their specificity to bind to MicF (Aim 3). Initial validation will be done in E. coli; however, MicF is highly conserved in ?-proteobacteria and thus results from this work can be extended to other important pathogens. We expect our designed biomolecules to increase susceptibility to a subset of antibiotics, thus reducing the impact of pathogenic bacteria on human health. Furthermore, the strategy outlined here is generalizable and can be extended to other RNA regulation mechanisms involved in antibiotic resistance.