High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism

Abstract

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets. The adaptation of a bacterial pathogen to the environments encountered during infection requires the wholesale remodeling of cellular physiology. One of the major changes encountered upon infection is nutritional, as the bacterium is forced to utilize compounds scavenged from the host. Mycobacterium tuberculosis has the unusual ability to use host cholesterol as a source of carbon and energy, and this capacity is required for persistence in animal models. However, our ignorance of the biochemical pathways involved in sterol degradation has limited our ability to assess the importance of this carbon shift in shaping the metabolic state of the bacterium. In this work, we developed a new method to quantitatively profile the fitness of thousands of mutants simultaneously. This allowed the identification of each bacterial gene that is required for the bacterium to grow using cholesterol as a carbon source. Reconstruction of the pathways comprised by these essential gene products revealed that adaptation to cholesterol required widespread metabolic changes. These genes account for a significant fraction of the bacterial functions important for growth in animal tissues, suggesting that the physiology of this intracellular pathogen is shaped by carbon sources available in this environment.