Generation of Diacylglycerol in Mycobacteria: Study of Key Enzymes for the Syn thesis of Triacylglycerides.

GRAMAJO, H; CROTTA ASIS, A; GAGO, G.; JACKSON, MARY

Banff

Congreso; Tuberculosis: Mechanisms, Pathogenesis and Treatment. Keystone Symposia; 2019

Keystone Symposia

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects one-third of world's population, producing the death of 2 million people per year. The pulmonary macrophages are the primary host cells of M. tuberculosis. During the first week of infection, the bacilli are able to replicate actively. When the host's immune system responses, lymphocytes are recruited at the site of infection and the infected macrophages die. This leads to the formation of the granuloma, a distinctive feature of infection with M. tuberculosis. Within the granuloma, M. tuberculosis may persist for decades, in a dormant state called ?latency?. One of the most striking features of the TB granuloma is the presence around the lesion of a specific population of macrophages enriched in lipid droplets known as foamy macrophages (FM). Within FM, M. tuberculosis decreases its multiplication rate and accumulates intracytoplasmic lipid inclusions (ILI) in its own cytoplasm which consist mainly of triacylglycerides (TAG). Furthermore, it is known that the reversion of the foamy phenotype leads to the progressive depletion of the accumulated ILI in bacterial cytoplasm and the resumption of mycobacterial division. However, the mechanisms by which M. tuberculosis induces the differentiation of these FM and by which ILI accumulates in their cytoplasm within infected cells are not known yet. This is due to the limited knowledge of the regulatory network involved in the maintenance of lipid homeostasis in mycobacteria, particularly in the regulation of TAG biosynthesis.The main biosynthetic pathway for TAG synthesis involves the sequential esterification of glycerol-3-phosphate to produce phosphatidic acid (PA). The PA is a key molecule in the synthesis of membrane glycerophospholipids through the synthesis of CDP-diacylglycerol. In M. tuberculosis, the PA can be dephosphorylated by a phosphatidic acid phosphatase enzyme (PAP) giving diacylglycerol (DAG), which is the direct precursor of TAG synthesis. Therefore, DAG synthesis is the first reaction specifically dedicated to the synthesis of TAG, suggesting a key role for the PAP enzyme in the regulation of PA flow towards the synthesis of TAG or membrane phospholipids. The main goal of our project is to elucidate the role of the key enzymatic step that governs the decision of M. tuberculosis to synthesize TAG and, therefore, slow its growth and enter dormancy. To accomplish this goal, we overexpressed two candidate proteins in M. smegmatis and found that both strains showed higher levels of DAG and consequently of TAG. Furthermore, we constructed mutant strains in these genes and studied their physiological role using a global approach that included lipidomics and proteomics. Our results suggest that both enzymes are involved in TAG synthesis and their depletion leads to a metabolic rearrangement that provides DAG to M. smegmatis by alternative pathways.