Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages

Abstract

Two billion people are latently infected with Mycobacterium tuberculosis ( Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 ( tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.

Author Summary

Two billion people are latently infected with Mycobacterium tuberculosis ( Mtb). Cure and possible eradication of tuberculosis are limited by the lack of availability of any drug that can kill dormant Mtb. Understanding of the processes critical for dormancy and a reliable dormancy model suitable for high throughput screening of chemicals will help to discover drugs that can kill dormant Mtb. Storage of lipids for utilization as energy source is critically needed for dormancy. In the human lung, Mtb-infected macrophages are sequestered inside the hypoxic environments of the physical enclosure called granuloma in which Mtb becomes dormant. None of the currently used cell culture models of Mtb infection mimic this situation. We developed a model that mimics the environment inside the human granuloma by incubating Mtb-infected macrophages under hypoxia. We found that, under these conditions, macrophages accumulate lipid droplets and Mtb within these macrophages acquire a dormancy phenotype. We report how the pathogen inside the macrophages utilizes the host lipids to store lipids within the pathogen and acquire the hallmark traits of dormant Mtb. Thus, our novel model of Mtb dormancy may enable better understanding of the metabolic processes vital for the dormant pathogen and help to discover drugs that can kill latent pathogens.