Project Summary Mycobacterium tuberculosis (Mtb) is the leading cause of death by infectious disease, with two million deaths annually. For Mtb to thrive within the host, the bacterium must obtain nutrients, including carbon sources which are essential for growth. Mtb can use four primary carbon sources to meet its needs for carbon utilization. Mtb primarily resides within the phagosome of host macrophages and must acquire carbon sources from the macrophage while in this niche. While in this compartment, Mtb also experiences a wide variety of stresses including reactive nitrogen species (RNS) stress. Previous work has shown that in vitro exposure of Mtb to nitric oxide (NO) leads to the nitrosylation of proteins that are essential for glycolysis. Additional preliminary data from our lab shows that either exposing Mtb transposon libraries to NO in vitro leads to an increased importance of fatty acid degradation and decreased reliance on glycolytic pathways during NO exposure. This has led us to hypothesize that exposure to RNS stress leads to changes in the use of carbon by Mtb. To begin to identify how RNS stress effects carbon utilization, Aim 1 will utilize a library of strains that are unable to either utilize or acquire each of the carbon sources. This library will be grown in the presence of single carbon sources and RNS, and growth of each strain will be monitored to determine how the RNS stress affects the ability of each mutant to grow on each carbon source. In Aim 2, this library will be used to infect macrophages and mice that are either wildtype or lack the ability to induce NO expression. These studies will allow us to determine how actual infection conditions affect the ability of Mtb to use various carbon sources. Finally, Aim 3 will focus on identifying the mechanisms by which RNS stress leads to changes in carbon usage by Mtb. These studies will provide important data on how Mtb utilizes carbon under stress conditions and the mechanisms which allow for changes in carbon utilization in vivo.