Generation of diacylglycerol in mycobacteria: study of key enzymes for the synthesis of triacylglycerides

GAGO, G; GRAMAJO, H; CROTTA ASIS, A

Congreso; XIII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2018

SAMIGE

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), infects one-third of world's population, producing the death of 2.4 million people per year. The pulmonary macrophages are the primary host cell 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 state of dormancy called ?latency?. The differentiation of macrophages into foamy macrophages (FM) is particularly important since they have been detected in patients who have developed reactivation of a primary TB infection. 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 foamy macrophages and by which ILI accumulates in their cytoplasm within infected cells are not known yet. This is due to the limited knowledge of the regulation 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 oleaginous bacteria like M. tuberculosis, the PA could be dephosphorylated by a phosphatidic acid phosphatase enzyme (PAP) giving as result 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. In the present work we demonstrate the phosphatase activity of three putative PAP enzymes of mycobacteria and analysed its physiological role, in order to characterize it at biochemical and genetic level. The main goal of our research 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.