Loss of Ag85A disrupts plasma membrane domains and promotes free mycolic acid accumulation in mycobacteria

Abstract

The mycomembrane of mycobacteria, composed primarily of long-chain mycolic acids, is critical for cell survival, structural integrity, and resistance to environmental stress, yet its underlying synthesis mechanisms remain incompletely understood. This study investigates the role of Ag85A, a key enzyme in mycomembrane synthesis, in regulating plasma membrane domains and cell envelope organization in Mycobacterium smegmatis. Using ΔAg85A deletion mutants, we combined microscopy, biochemical assays, thin-layer chromatography, and lipid analysis to evaluate changes in membrane structure, chemical accumulation, and lipid composition. Ag85A deletion leads to altered plasma membrane domain organization, increased chemical accumulation, changes in cell envelope lipid composition. Unexpectedly, lipid analysis revealed accumulation-not depletion-of mycolic acids in the mutant, suggesting that increased permeability is not directly due to mycolic acid loss. These findings highlight a novel link between mycomembrane composition and plasma membrane domain stability. Our study not only advances understanding of mycobacterial cell envelope architecture but also identifies potential targets for enhancing drug penetration in resistant mycobacterial infections.

Keywords: Antigen 85A; Membrane domain; Membrane permeability; Mycobacteria; Mycolic acids.

Figure 1.

Ag85A protects M. smegmatis from the membrane fluidizer accumulation. (A) Left, the survival of ΔAg85A after 3-hour treatment of water or dibucaine treatment was analyzed on the 7H10 plate. Right, colony-forming units (CFU) were calculated from three biological triplicates. There were no statistically significant differences in wild-type (indicated as ns) but ΔAg85A showed the statistical differences (* P<0.05) as determined by Mann-Whitney U test. (B) Dibucaine accumulation in cell envelope was quantified by liquid chromatography-mass spectrometry. p-values were determined by KruskalKruskal-Wallis test with Dunn's multiple comparisons test from three technical replicates from three biological replicates.replicates ns p=0.4936, p=0.0323 (C) Cells were pretreated with either ethambutol or DMSO (vehicle control) for 3 hours. After the pre-incubation, the cells were washed then treated with dibucaine or water for an additional 3 hours. CFU counts were obtained for each group both before and after dibucaine treatment, and CFU ratios were calculated by dividing the CFU of the dibucaine-treated group by the CFU of the water-treated group. P=values were determined by Mann-Whitney U test. * p=0.0470.

Figure 2.

The depletion of Ag85A induced IMD delocalization. (A) Delocalization of mCherry-GIfT2 by the depletion of Ag85A in M. smegmatis. mCherry-GIfT2 was imaged after 12-hour anhydrotetracycline treatment. Images are representative of three independent experiments, and fluorescence distributions of the fusion proteins after chemical treatment were calculated from three independent experiments. Lines show the average of all cells (50 < n < 75). Signal was normalized to cell length and total fluorescence intensity. Scale bar, 5 μm. (B, left) Diagram of IMD and PM-CW fractionation after ultracentrifugation. (B, right) To visualize IMD and PM-CW, PimB’ and MptC which are established marker protein were analyzed by western blotting. Asterisks indicate the proteins analyzed.

Figure 3.

Mycolic acid and unknown glycolipid are accumulating in outer membrane of ΔAg85A. (A) TMM and TDM were accumulated in ΔAg85A. TMM and TDM were resuspended in chloroform, methanol, and water (9:1:0.1, v/v/v), chromatographed using chloroform, and visualized by orcinol. (B) Mycolic acid and fatty acid were methylated and chromatographed using a solvent containing petroleum ether and acetone (95:5, v/v). (C) Phospholipids 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 molybdenum blue (Rf = 0.35–0.61). (D) PIMs from the indicated mutants were separated as in the panel C (Rf = 0.20–0.48) and visualized by orcinol. mmpl3 depletion strain was used for controls which accumulate mycomembrane precursor (TMM) in the plasma membrane. CL, cardiolipin; and PE, phosphatidylethanolamine. Pl, phosphatidylinositol. PIM, phosphatidylinositol mannoside. All experiments were repeated at least twice. Bands were assigned as the expected molecules based on migration patterns reported in previous literature.

Figure 4.

The unknown glycolipid is stained by iodine and susceptible to alkaline hydrolysis. (A) The unknown glycolipid was 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 iodine vapor. (B) Lipid extracts were treated with 0.2M NaOH and resuspended in chloroform, methanol, and water (10:10:3, v/v/v). The plate was predeveloped by chloroform and developed by chloroform, methanol (9:1, v/v). Orcinol was used for visualization.