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.

We isolated a rough variant of Mycobacterium abscessus CIP 104536T during experimental infection of mice. We show that this variant has lost the ability to produce glycopeptidolipids, is hyperlethal for C57BL/6 mice infected intravenously, and induces a strong tumor necrosis factor-alpha response by murine monocyte-derived macrophages.Mycobacterium abscessus (formerly Mycobacterium chelonae subsp. abscessus) is an emerging, rapidly growing mycobacterium that causes a wide spectrum of human infections, including skin and soft tissue infections (3, 26), lung infections (11, 24), and disseminated infections in patients either under immunosuppressive therapy (3, 23) or with a Mendelian syndrome conferring susceptibility to mycobacteria (5). Extended lung infections and disseminated infections raise serious therapeutic issues because M. abscessus strains are resistant to most antibiotics and are associated with a particular high fatality rate (3,23).M. abscessus organisms may be isolated with a smooth (S) or a rough (R) morphotype from clinical samples (23). Recently, Byrd and Lyons described an R strain of M. abscessus from an ileal granuloma in a patient with Crohn's disease, and they reported the spontaneous in vitro dissociation of this isolate into an S variant (4). Interestingly, these authors showed that the S variant was severely attenuated in its ability to infect the murine host and to persist in human monocytes. M. abscessus may thus undergo an S/R phase variation linked to pathogenicity. The molecular basis for the S or R appearance of M. abscessus, as well as the reason for the difference in pathogenicity of S and R variants, is presently unknown (12).We have recently used M. abscessus CIP 104536T (ϭATCC 19977T) (19) to study the immune control of M. abscessus infection in the murine host. This strain, which was provided by the Laboratoire de Référence des Mycobactéries (Institut Pasteur, Paris, France) has an S morphotype on ordinary solid media such as Trypticase soy agar (BioMérieux, Marcy l'Etoile, France) (Fig. 1A). Following the intravenous (i.v.) injection of 10 7 CFU of CIP 104536T into immunoglobulin chain knockout mice (15), we obtained mycobacterial colonies of the R morphotype from deep organs of several animals on day 90 following infection. We verified that these colonies were truly M. abscessus by partial hsp65 sequencing (21). One isolated colony (CIP 104536T-R) was selected, reisolated, and cryopreserved at Ϫ80°C using cryobeads (Mast Diagnostics, Reinfeld, Germany). The R morphotype of this variant was confirmed to be stable after 10 subcultures on solid media and three iterative in vivo passages. Thus, in contrast with previous studies reporting the switch of an R M. abscessus strain into an S morphotype (4), we show here that R variants may be selected in vivo from an M. abscessus strain with an S morphotype. Glycopeptidolipids (GPLs), also called C-mycosides or Jsubstances, represent up to 85% of the surface-exposed lipids in M. abscessus and a number of other nontuberculous mycobact...