Abstract
The mycobacterial envelope is unique, containing the so-called mycomembrane (MM) composed of very-long chain fatty acids, mycolic acids (MA). Presently, the molecular composition of the MM remains unproven, due to the diversity of methods used for determining its composition. The plasma membranes (PM) and the native MM-containing cell walls (MMCW) of two rapid-growing mycobacterial species, Mycobacterium aurum and M. smegmatis, were isolated from their cell lysates by differential ultracentrifugation. Transmission electron microscopy and biochemical analyses demonstrated that the two membranes were virtually pure. Bottom-up quantitative proteomics study indicated a different distribution of more than 2,100 proteins between the PM and MMCW. Among these, the mannosyltransferase PimB, galactofuranosyltransferase GlfT2, Cytochrome p450 and ABC transporter YjfF, were most abundant in the PM, which also contain lipoglycans, phospholipids, including phosphatidylinositol mannosides, and only a tiny amount of other glycolipids. Antigen85 complex proteins, porins and the putative transporters MCE protein family were mostly found in MMCW fraction that contains MA esterifying arabinogalactan, constituting the inner leaflet of MM. Glycolipids, phospholipids and lipoglycans, together with proteins, presumably composed the outer leaflet of the MM, a lipid composition that differs from that deduced from the widely used extraction method of mycobacterial cells with dioctylsulfosuccinate sodium.
Highlights
Mycobacteria are probably the most successful microorganisms to parasite animals and humans
These results suggested that the M. aurum (Mau) F2 fraction corresponded to the MM-containing cell wall (CW) (MMCW) whereas the Mau F1 fraction was assignable to the plasma membrane (PM), and that the former fraction was virtually free from significant contamination by the latter one
The central role of the mycobacterial cell envelope in essential physiological processes makes the determination of its composition and arrangement of its constituents an important field of research
Summary
Mycobacteria are probably the most successful microorganisms to parasite animals and humans. The mycobacterial cell wall contains up to 60% of lipids, as compared with some 20% for the lipid-rich cell walls of Gram-negative microorganisms[4] These lipids include the exceptionally-long chain fatty acids (mycolic acids, MA) covalently linked to the cell wall polysaccharide arabinogalacatan (AG) and whose esterifying trehalose, as well as the numerous classes of exotic compounds typifying the Mycobacterium genus. Others and we have demonstrated that the MM thickness is around 7–8 nm[6,8] despite the presence of the very long chain of MA (up to C90), which raises important questions about the exact native conformation of MA This outer membrane is supposed to contain porins for the uptake of small hydrophilic molecules – porins characterized only in the case of M. smegmatis (Msm)[16] and membrane machineries involved in the secretion of virulence factors such as the well described ESAT-6 and Cfp[10] proteins[17,18,19], whose mechanism of translocation across the MM remains largely unknown. As a proof of the principle, we combined various methods to isolate, for the first time, the MMCW of two mycobacterial model species, namely M. aurum (Mau) and Msm, virtually free from significant contamination by the PM and identify the lipids constitutive of the mycomembrane
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