Abstract Mycobacterium tuberculosis (Mtb) is a human pathogen that causes significant morbidity and mortality worldwide. An increasing number of tuberculosis cases are caused by multi-drug and extreme-drug resistant Mtb strains, underscoring the need for novel therapeutics. A hallmark of tuberculosis is that Mtb manipulates the host to allow for latent infections. Latency is mediated by the stringent response via production of (p)ppGpp signaling nucleotides by the Mtb enzyme relA. This was demonstrated by the failure of Mtb relA mutants to sustain a chronic infection phase in the lungs of mice or guinea pigs. In addition, the stringent response is associated with persister status, which confers antibiotic resistance in bacteria. Despite this well-established importance of the stringent response, to date no (p)ppGpp-binding proteins have been identified in Mtb and consequently the molecular mechanisms of (p)ppGpp-mediated signaling are poorly understood. Here we propose the hypothesis that Mtb expresses (p)ppGpp binding proteins that will be identified via a well established, high-throughput and unbiased screening approach. In AIM1 we plan to generate an MBP- and His-tagged Mtb ORFeome expression library in E. coli using an existing Gateway ORFeome library. This library will be screened for (p)ppGpp-binding proteins using the DRaCALA assay. In AIM2 we will use a conditional relA-expressing Mtb strain to characterize the (p)ppGpp regulon via RNAseq. In addition, we will create and analyze Mtb deletion mutants in identified (p)ppGpp-binding proteins. The identification of novel mediators of the stringent response in Mtb will generate a better molecular understanding of the pathways mediating latency in Mtb and these findings will generate novel targets for therapeutic development against Mtb.

Narrative Mycobacterium tuberculosis causes tuberculosis in about 10 million people annually leading to 1-2 million deaths. The current research project aims to identify (p)ppGpp-binding proteins of Mtb and characterize their importance for virulence mechanisms of the bacterium. The results of this project could be exploited for new drug designs and improved vaccine generation.