The rapid rise of multidrug-resistant (MDR) superbugs and the declining antibiotic pipeline are serious challenges to global health. Rational design of antibiotics can accelerate development of effective therapies against MDR bacteria. In this talk, I will describe multi-pronged systems and synthetic biology based approaches being devised in our lab to rationally engineer therapeutics that can overcome antimicrobial resistance by controlling gene expression. Transcriptome studies in our lab have shown that, when exposed to antimicrobials, bacteria enter an “adaptive resistance” state by exploring multiple pathways sampling a dynamic gene regulatory space. We have developed an approach dubbed “Controlled Hindrance of Adaptation of OrganismS” or “CHAOS” to slow the evolution of antibiotic resistance by interfering with processes involved in adaptive resistance. Using CRISPR based technology, we rationally engineer library of synthetic genetic devices for multiplexed activation and inhibition of native gene expression of key essential and stress-response gene networks. Here we show that CHAOS approach leads to predominant negative epistasis with severe loss of fitness during adaptation to a range of antibiotics and eventual “slowing” down of bacteria's ability to adapt. We also show how bacteria may be using the mechanism of Transcriptional interference (TI) to generate different levels of gene expression and variability. TI occurs when a transcriptional process is negatively affected by the presence of neighboring RNA polymerase traffic or proteins. Given that a large number of bacterial stress response genes are physically arranged in tandem or convergent orientation, TI offers a promising mechanism for tunable gene regulation. We demonstrate that by engineering the extent of TI, gene expression response can be modified easily to manipulate the overall regulatory range, sharpness of the induction curve, expression variability, as well as creation of higher-order logic gate behavior. Such behaviors can be successfully described by a mathematical model of TI, presenting the opportunity to both understand how naturally occurring systems integrate multiple inputs exerted by transcription. Finally, to translate our findings into the clinical setting, we are developing antisense therapeutics that can block translation of any desired gene in a pathogen-specific manner for targeted inhibition. We are building a Facile Accelerated Specific Therapeutic (FAST) platform for the accelerated development of peptide nucleic acid (PNA) based novel antibiotics against MDR bacterial clinical isolates as well as any emergent bacterial threats. By regulating gene expression FAST can eliminate a broad range of MDR bacterial clinical isolates including methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase producing Klebsiella pneumoniae and Salmonella typhimurium, and carbapenem-resistant Escherichia coli. The gene expression perturbation platforms presented in this talk offer novel approaches for impeding the evolution of antibiotic resistance, developing new antibiotics, as well as re-sensitizing antibiotic-resistant pathogens to traditional therapies employed in the clinical setting.

This work is supported by DARPA Young Faculty award (D17AP00024) and National Science Foundation (MCB1714564).