![]() ![]() DMSO, dimethyl sulfoxide GPL, glycopeptidolipid HPTLC, high-performance thin layer chromatography PIM, phosphatidylinositol mannoside TDM, trehalose dimycolate.Mycobacteria cause a number of dangerous, difficult-to-treat diseases including leprosy and tuberculosis, and progress has been slow in eradicating them. All experiments were repeated at least twice, and representative results are shown. CL, cardiolipin and PE, phosphatidylethanolamine. C–E: HPTLC analysis of lipids: (C) PIMs separated and visualized as in panel B (Rf = 0.20–0.48) (D) phospholipids separated as in panel B and visualized by molybdenum blue (Rf = 0.35–0.61) (E) GPLs and TDM separated by chloroform/methanol/water (9:1:0.1, v/v/v) and visualized by orcinol. A region of the HPTLC plate corresponding to Rf of 0.22–0.48 is shown. PIMs were chromatographed using a solvent containing chloroform, methanol, 13 M ammonia, 1 M ammonium acetate, and water (180:140:9:9:23, v/v/v/v/v) and visualized by orcinol staining. B: HPTLC analysis of PIMs after heat killing followed by benzyl alcohol treatment. A: Live and heat-killed cells were treated with benzyl alcohol for 60 min, serially diluted, and plated onto Middlebrook 7H10 agar, confirming that rapid heating is effective in killing M. smegmatis. All rights reserved.Įffect of benzyl alcohol on M. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.Ĭell envelope enzyme regulation glycolipids heat stress lipids membrane fluidity metabolism pathogens plasma membrane integrity stress response.Ĭopyright © 2022 The Authors. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol pre-exposure. Additionally, we show that PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Upon membrane fluidization, we determined the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. Without membrane stress, PIMs are predominantly in a triacylated form: two acyl chains of the PI moiety plus one acyl chain modified at one of the mannose residues. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) are rapidly remodeled upon membrane fluidization in Mycobacterium smegmatis. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of the plasma membrane to stress exposure remain poorly understood. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. ![]()
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