Elucidating the link between Mycobacterium tuberculosis potassium homeostasis and its lipid metabolism and growth in vivo

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PROJECT SUMMARY/ABSTRACT Successful host colonization by Mycobacterium tuberculosis (Mtb) requires that the bacteria sense and respond to disparate environmental cues coordinately during infection, and adapt its physiology accordingly. This includes an ability to respond to acidic pH and maintain intrabacterial ionic homeostasis, as well as utilization of lipid carbon sources during infection. However, how Mtb ionic cue response and homeostasis and its lipid metabolism are functionally connected remains poorly understood. Intriguingly, we have found that Mtb lipid metabolism and its potassium (K+) response/homeostasis is linked. For example, the bacterial response to cholesterol is repressed upon disruption of intrabacterial K+ homeostasis, and growth in cholesterol media of a Mtb mutant disrupted in K+ homeostasis is impeded at pH 7, but not pH 6. Aim 1 of this proposal thus seeks to define the global impact of Mtb K+ homeostasis disruption on the bacterium’s ability to utilize cholesterol, examining both transcriptional response and utilizing a fluorescent cholesterol tracer for the analysis of cholesterol uptake. Aim 2 focuses on understanding the functional impact of local environmental differences on intrabacterial K+ homeostasis and Mtb replication status during infection, with single bacterium resolution. It will exploit the use of a novel fluorescent reporter that reports on intrabacterial K+ levels, with tests on wild type Mtb and mutants in several key lipid utilization regulators. Single bacterium level analysis in vivo is particularly critical due to the marked heterogeneity in both local pH and nutrient sources experienced by Mtb during infection, which extends to non-uniformity spatially within canonical caseous necrotic lesions. As such, Aim 2 studies will further exploit combination of a fluorescent replication reporter with a Mtb mutant disrupted for intrabacterial K+ homeostasis, in a murine infection model that recapitulates highly structured caseous necrotic lesions observed during human infection. These in vivo experiments will enable elucidation of how differences in local environment (pH, carbon source etc.) during infection may alter the relative need for active maintenance of intrabacterial K+ homeostasis by Mtb for replication success. This project is conceptually innovative in examining how Mtb K+ homeostasis affects its lipid metabolism in a pH-dependent manner, an intersection of vital aspects of Mtb infection biology that is unexplored. There is further methodological innovation in the development of a new intrabacterial [K+] reporter to delineate the impact of local environment in vivo on Mtb K+ homeostasis. These studies will provide the foundation for understanding the concept of an intrinsic link between Mtb metabolism and ionic cue response and homeostasis, a facet of Mtb infection biology that holds the potential for exploitation for therapeutic purposes.