Glutamine Starvation as a Means to Enhance Antibiotic Efficacy against M. tuberculosis

Project Summary Mycobacterium tuberculosis (Mtb) is a public health threat responsible for nearly 2 million deaths annually. While an efficacious chemotherapeutic regimen exists, infection relapse is common even after an extensive treatment time (6-9 months). Recent evidence indicates that Mtb antibiotic tolerance directly impacts treatment times and opens the window for the selection of antibiotic resistance in persistent bacterial populations. Multiple studies have shown that heterogeneously expressed immune pressures such as reactive nitrogen species induce antibiotic tolerance in Mtb sub-populations. Therefore, one strategy to improve antibiotic efficacy is to target pathways that reduce Mtb antibiotic tolerance in the face of immune insult. Data from the Sassetti lab and others indicated that loss of Mtb glutamine (Gln) import or synthesis reduces bacterial viability and antibiotic tolerance during murine infection. Based on these findings, the hypothesis of this proposal is that Gln starvation leaves Mtb vulnerable to host immune pressures which translates as reduced antibiotic tolerance and increased drug efficacy in vivo. Therefore, this proposal seeks to understand the mechanistic link between Gln starvation, immune pressure vulnerability, and reduced antibiotic tolerance. To do this, the experiments described will use Mtb strains deficient for Gln synthesis and/or host Gln import as a means to induce Mtb Gln starvation and disrupt Gln metabolism. These strains will be used to assess how Gln starvation impacts antibiotic efficacy during murine and intracellular infection. Aim 1 of this proposal will use single cell RNAseq to probe Mtb heterogeneity during murine infection and treatment. Aim 2 will explore the mechanistic links between immune pressures such as reactive oxygen species (ROS), reactive nitrogen intermediates (RNI), phagosome acidification, Gln restriction and Mtb antibiotic tolerance using engineered macrophage cell lines. Finally, as a proof of concept, Aim 3 will use the relapsing mouse model to assess the impact Gln starvation has on infection clearance and relapse following an extended antibiotic treatment. The PIs assert that the data generated in this proposal will pave the way for new therapeutic strategies that both directly target Mtb viability and disrupt antibiotic tolerance leading to a more efficacious therapeutic regimen. The research plan proposed will act as a means to expand on the trainee’s skill set with a new emphasis on host-pathogen interactions. The techniques proposed include novel host and bacterial genetic approaches, high content murine immune profiling coupled with infected cell sorting, and evaluation of antibiotic efficacy in multiple animal models. The training environment described in this proposal provides the trainee with opportunities to train under senior researchers with expertise in host and bacterial genetics, high throughput cell profiling, and high content sequencing analysis. The completion of this proposal will establish the trainee in methods with techniques to become an independent scientist.