Author: Dr. Jaiyanth Daniel Assistant Professor, Department of Biology Indiana University - Purdue University Fort Wayne
Mycobacterium tuberculosis (Mtb) causes latent tuberculosis infection in one-third of the world population and remains quiescent in the human body for decades. Dormant Mtb becomes phenotypically tolerant to antibiotics, loses acid fastness and accumulates triacylglycerol (TAG)-containing lipid droplets for possible use as energy source during dormancy and reactivation. The phenotypic drug tolerance of dormant Mtb necessitates a prolonged treatment regimen which causes poor compliance and contributes to the emergence of multi-drug resistance. The dormant pathogen accumulates lipid droplets containing TAG which provides metabolic energy for use during its dormant state within the human body. Therefore, we are investigating the metabolism of fatty acids for the biosynthesis of TAG by Mtb and seek to understand the roles of Mtb genes involved in such processes. Our studies could provide insights that enable the development of novel therapies that target the dormant pathogen inside the human body and shorten the time required for curing tuberculosis.
In mammals, perilipin regulates lipid droplet homeostasis but no such protein has been identified in Mtb. We identified an Mtb protein, MPER1, that showed weak amino acid sequence identities with mammalian perilipin-1 and was upregulated in Mtb dormancy. We generated a ppe15 gene-disrupted mutant of Mtb and examined its ability to metabolically incorporate radiolabeled oleic acid into TAG, accumulate lipid droplets containing TAG and develop phenotypic tolerance to rifampicin in two in vitro models of dormancy including a three-dimensional human granuloma model. The mutant showed a significant decrease in the biosynthesis and accumulation of lipid droplets containing TAG (Figure 1). Our findings suggest that the MPER1 protein plays a critical role in the homeostasis of TAG-containing lipid droplets in Mtb and influences the entry of the pathogen into a dormant state.
Figure 1. The Mtb mutant (d-mper1) lacking the mper1 gene was imparied in lipid droplet formation (A) and TAG biosynthesis (B, C) under dormancy-inducing conditions, compared to the wild-type (WT). TAG, Triacylglycerol; PL, polar lipids. Figure from Daniel et al. 2016.
Mtb acquires fatty acids from the human body and these fatty acids are modified by acyl-coenzyme A synthetases in Mtb prior to being utilized in metabolic pathways. Since no such enzyme had been shown to be involved in TAG metabolism in Mtb, we investigated the functions of the Mtb gene Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins. We demonstrated that it is able to stimulate fatty acid uptake in E. coli cells. Purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in lipids in the human body. Our results indicate that the expression of FACL6 protein in Mtb is significantly increased during in vitro dormancy. The facl6-deficient Mtb mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function (Figure 2). Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids.
Figure 2. FACL6 is an acyl-coenzyme A synthetase. A, Purified FACL6 synthesizes acyl-CoA in a CoA- and ATP-dependent manner. B, Quantification of radioactive acyl-CoA bands in A. The FACL6 protein stimulates fatty acid uptake in E. coli (C). Deletion of FACL6 inhibits triacylgylcerol synthesis in M. tuberculosisduring in vitro dormancy (D). E, Quantitation of radioactive acyl-CoA bands in D. Figure from Daniel et al. 2014
Daniel, J†, N. Kapoor, T. Sirakova, R. Sinha & P. Kolattukudy, (2016) The perilipin-like PPE15 protein in Mycobacterium tuberculosis is required for triacylglycerol accumulation under dormancy-inducing conditions. Mol Microbiol 101: 784-794. †Corresponding Author.
Daniel, J†, Sirakova, T. & Kolattukudy, P. (2014) An Acyl-CoA Synthetase in Mycobacterium tuberculosis Involved in Triacylglycerol Accumulation during Dormancy. PLoS One 9(12):e114877. †Corresponding Author.