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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.

SUBMITTER: Griffin JE

PROVIDER: S-EPMC3182942 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.

Griffin Jennifer E JE Gawronski Jeffrey D JD Dejesus Michael A MA Ioerger Thomas R TR Akerley Brian J BJ Sassetti Christopher M CM

PLoS pathogens 20110929 9

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 mutagenesi ...[more]

PMID: 21980284

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Project description:Cholesterol of the host macrophage membrane is vital for mycobacterial infection, replication, and persistence. During chronic infection within host lung tissues, cholesterol facilitates the phagocytosis of mycobacteria into macrophages. Cholesterol degradation leads to increased ﬂux of acetyl-coenzyme A (CoA) and propionyl-CoA, providing energy and building blocks for virulence macromolecules as well as donors for global protein acylation. Potential functions of lysine acylation are gradually revealed in bacterial survival and pathogenesis. However, the mycobacterial proteome and posttranslational modification (PTM) changes involved in the cholesterol catabolism bioprocess remain unclear. Here, we used nonpathogenic Mycobacterium smegmatis as a model and simultaneously monitored mycobacterial proteome and acetylome changes in the presence of glucose and cholesterol. We discovered that cholesterol metabolic enzymes were upregulated with respect to both protein expression levels and lysine acylation levels during the metabolic shift from glucose to cholesterol. After that, adenylating enzymes related to cholesterol metabolism were proven to be precisely regulated at the propionylation level by mycobacterial acyltransferase M. smegmatis Kat (MsKat) in response to cellular propionyl-CoA accumulation. Furthermore, the kinase expression and phosphorylation levels were also changed along with fluctuations in cholesterol levels. Our results expanded current knowledge of acylation regulation in the cholesterol catabolism of mycobacteria and provided references for possible antimycobacterium strategy.IMPORTANCE Cholesterol assimilation is a critical step in mycobacterial chronic infection. However, knowledge from the dynamic characterization of cholesterol metabolism in mycobacteria at the protein expression and PTM levels remains limited. Our study uncovered the landscape of protein expression, lysine acetylation, lysine propionylation, and S/T/Y phosphorylation during the metabolic changes from glucose to cholesterol in mycobacteria. The data showed that cholesterol-induced carbon shift resulted in the elevation of protein expression and lysine acylation in diverse metabolic enzymes involved in cholesterol degradation and that the presence of cholesterol also promoted the perturbations at the phosphorylation level in the kinase system in mycobacteria. This study systematically characterized the regulation of cholesterol catabolism at several different levels, which provided the detailed references in mycobacterial proteome and potential antimycobacterial strategies.

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High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.

Publications

High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.

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