Abstract
Tuberculosis caused by Mycobacterium tuberculosis is currently one of the leading causes of death from an infectious agent. The main difficulties encountered in eradicating this bacteria are mainly related to (i) a very complex lipid composition of the bacillus cell wall, (ii) its ability to hide from the immune system inside the granulomas, and (iii) the increasing number of resistant strains. In this context, we were interested in the Rv0646c (lipGMTB) gene located upstream to the mmaA cluster which is described as being crucial for the production of cell wall components and required for the bacilli adaptation and survival in mouse macrophages. Using biochemical experiments combined with the construction of deletion and overexpression mutant strains in Mycobacterium smegmatis, we found that LipGMTB is a cytoplasmic membrane-associated enzyme that displays both phospholipase and thioesterase activities. Overproduction of LipGMTB decreases the glycopeptidolipids (GPL) level concomitantly to an increase in phosphatidylinositol (PI) which is the precursor of the PI mannoside (PIM), an essential lipid component of the bacterial cell wall. Conversely, deletion of the lipGMS gene in M. smegmatis leads to an overproduction of GPL, and subsequently decreases the strain susceptibility to various antibiotics. All these findings demonstrate that LipG is involved in cell envelope biosynthesis/remodeling, and consequently this enzyme may thus play an important role in mycobacterial physiology.
Highlights
With more than 10 million new cases and almost 1.6 million deaths in 2017, tuberculosis (TB) caused by the etiologic agent Mycobacterium tuberculosis (M. tuberculosis) still remains the deadliest infectious disease worldwide [1]
In M. tuberculosis, Rv0646c is surrounded by an essential uncharacterized gene (Rv0647c) and the mmaA cluster composed of four mmaA genes encoding proteins involved in mycolic acid (MA) maturation processes (Figure 1)
The occurrence and genome organization of lipG clusters are highly conserved in M. tuberculosis, M. leprae, M. marinum, or M. smegmatis genomes (Figure 1), with two copies of genes encoding for LipG proteins in M. marinum, M. abscessus, or M. ulcerans genomes
Summary
With more than 10 million new cases and almost 1.6 million deaths in 2017, tuberculosis (TB) caused by the etiologic agent Mycobacterium tuberculosis (M. tuberculosis) still remains the deadliest infectious disease worldwide [1]. Amongst the eight putative methyltransferases identified in M. tuberculosis H37Rv genome, the mmaA gene cluster; i.e. mmaA1 (Rv0645c), mmaA2 (Rv0644c), mmaA3 (Rv0643c), and mmaA4 (Rv0642c); is essential for chemical group introduction and functionalization of Mas, participating actively in the envelope integrity/virulence of M. tuberculosis [14,19,20] These mmaA genes are clustered with the Rv0646c gene, which is highly conserved amongst mycobacterial species and encodes for LipG (LipGMTB), a putative lipase/esterase which has been recently described as a carboxylesterase [21]. Due to its high conservation and its peculiar location upstream to the mmaA cluster, which is crucial for the production of cell wall components and so for the mycobacterial envelope integrity, the potential physiological function of LipG deserves to be studied and deciphered In this context, Rv0646c gene from M. tuberculosis H37Rv has been cloned, expressed, purified, and fully biochemically characterized. Subcellular location, in silico modeling, lipid extraction followed by TLC analysis, and susceptibility testing via the resazurin microtiter assay (REMA), we determined that LipGMTB is a cytoplasmic membrane-associated enzyme involved in phospholipids remodeling and cell wall integrity
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