Riboflavin Synthesis and Transport in Mycobacterium tuberculosis

PROJECT SUMMARY / ABSTRACT Metabolic pathways that Mycobacterium tuberculosis (Mtb) requires for growth represent promising targets for development of novel antibiotics to shorten tuberculosis (TB) treatment and combat multidrug resistant TB. Mtb requires the de novo riboflavin synthesis metabolic pathway for growth in vitro, but whether Mtb requires riboflavin synthesis during infection or can scavenge sufficient riboflavin from host tissues has not been directly tested. Knowledge of the Mtb riboflavin synthesis and transport mechanisms and their importance during infection will be critical to exploit these pathways for development of new TB drugs. The overall objective of this proposal is to define the importance of riboflavin synthesis and transport to Mtb survival in the host. The central hypothesis is that Mtb requires de novo riboflavin synthesis for replication and/or survival during all stages of mammalian infection, despite the existence of dedicated riboflavin transporters, because it cannot acquire sufficient riboflavin from host tissues. Preliminary data using a Mtb strain that conditionally expresses RibA2, which catalyzes the first step of riboflavin synthesis, provide compelling support for this hypothesis, which will be tested in three specific aims. Aim 1 will use the RibA2 conditional expression strain to determine the impact of riboflavin starvation on Mtb physiology and drug susceptibility and develop a panel of Mtb strains with hypomorphic expression of riboflavin synthesis enzymes to enable future drug discovery efforts. Aim 2 will determine the extent to which Mtb requires riboflavin synthesis in macrophage and mouse infection models using the RibA2 conditional expression strain. Aim 3 will use a synthetic lethal genetic screen to identify Mtb riboflavin transporters. The approach uses innovative genetic strategies to identify mechanisms of riboflavin transport and to identify synergy between riboflavin synthesis inhibition and existing anti-TB drugs. The proposed research is significant because it is expected to establish riboflavin synthesis as a viable target for development of new anti-TB drugs by demonstrating that riboflavin synthesis inhibition kills Mtb in mammalian infection models and sensitizes Mtb to existing antibiotics. The proposal will also lay the foundation for high throughput screens to identify small molecule riboflavin synthesis inhibitors with antibiotic activity.