Arginine Biosynthesis: A Metabolic Battery for Mycobacteria tuberculosis to Survive Aerosolization Stress - PROJECT SUMMARY Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the leading cause of death amongst infectious causes. While we have made progress reducing TB mortality, disease burden has stagnated due to our inability to reduce the incidence rate of TB. This can be attributed to our lack of transmission blocking interventions and the highly infectious nature of Mtb’ s aerosol transmission. Aerosolization represents an essential physiologic process for Mtb’ s transmission and infectious life cycle that can be targeted to reduce TB transmission and incidence. Mtb has likely evolved an adaptive response to the dramatic microenvironment changes it experiences as it transitions from the nutrient-rich lung to the desolate atmosphere. The specific genes and metabolic pathways that Mtb employs to navigate these changes are poorly understood. In the aerosol droplet environment, Mtb is deprived of CO2 and nitrogen sources which provide the carbon and nitrogen building blocks on which Mtb relies for biosynthetic processes. My project seeks to understand metabolic mechanisms underlying Mtb’ s response to CO2 and nitrogen deprivation. Preliminary data from our lab has identified arginine biosynthesis as a key metabolic pathway mediating Mtb’ s response to CO2 limitation. I propose that arginine biosynthesis mediates Mtb survival in the CO2 and nitrogen limited aerosol droplet environment, by maintaining a steady flux of carbamoyl phosphate, thermodynamically activated and short-lived essential intermediate. In this model, I propose that arginine biosynthesis is configured as a cycle mediated by ArcA or Rv2323c, a currently uncharacterized protein which I hypothesize to act as an arginine dihydrolase. Through this configuration, arginine biosynthesis couples NH4+ regeneration in ArcA or Rv2323c catalysis with the oxidative production of CO2 in the tricarboxylic acid (TCA) cycle. I hypothesize that Mtb can regulate between Rv2323c and ArcA based on nutrient availability. I predict that Mtb preferentially uses ArcA during CO2 and nitrogen limitation, since ArcA can mediate an arginine cycle without consuming CO2 and NH4+ equivalents. I will test this hypothesis with three Aims. In Aim 1, I will identify the biochemical role of Rv2323c in arginine metabolism through in vitro and in vivo characterization of Rv2323c’s catalytic activity and physiologic function. In Aim 2, I will characterize the roles of ArcA and Rv2323c in CO2 limited environments and measure their contributions to the endogenous pool of CO2 and impact on Mtb survival in air. In Aim 3, I will characterize the role of ArcA and Rv2323c in nitrogen limited environments by determining Mtb’ s preference for either enzyme in these conditions and their impact on Mtb’ s survival. These approaches will reveal novel insights into Mtb’ s adaptive response to aerosolization with the potential of identifying therapeutic targets to block Mtb transmission.
