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...

Glycopeptidolipids (GPLs) are a class of species-or type-specific mycobacterial lipids and major constituents of the cell envelopes of many non-tuberculous mycobacteria. To determine the function of GPLs in the physiology of these bacteria, a mutant of Mycobacterium smegmatis in which the gene encoding a mycobacterial nonribosomal peptide synthetase has been inactivated by transposon mutagenesis was analysed. Labelling experiments indicated that half of the bacterial GPLs were located on the cell surface and represented 85 % of the surface-exposed lipids of the parent strain whereas the mutant was defective in the production of the GPLs. Compared to the parent smooth morphotype strain, the GPL-deficient mutant strain exhibited a rough colony morphology, an increase of the cell hydrophobicity and formed huge aggregates. As a consequence, the mutant cells were no longer able to bind ruthenium red, as observed by transmission electron microscopy. The altered surface properties of the mutant cells also affected the phagocytosis of individual bacilli by human monocyte-derived macrophages since mutant cells were internalized more rapidly than cells from the parent strain. Nevertheless, no specific release of surface constituents into the culture broth of the mutant was observed, indicating that the cell surface is composed of substances other than GPLs and that these are essential for maintaining the architecture of the outermost layer of the cell envelope. Importantly, the absence of these major extractable lipids of M. smegmatis from the mutant strain has a profound effect on the uptake of the hydrophobic chenodeoxycholate by cells, indicating that GPLs are involved in the cell wall permeability barrier of M. smegmatis. Altogether, these data showed that, in addition to being distinctive markers of numerous mycobacterial species, GPLs play a role in the bacterial phenotype, surface properties and cell wall permeability.

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BackgroundThe outermost layer of the bacterial surface is of crucial importance because it is in constant interaction with the host. Glycopeptidolipids (GPLs) are major surface glycolipids present on various mycobacterial species. In the fast-grower model organism Mycobacterium smegmatis, GPL biosynthesis involves approximately 30 genes all mapping to a single region of 65 kb.ResultsWe have recently sequenced the complete genomes of two fast-growers causing human infections, Mycobacterium abscessus (CIP 104536T) and M. chelonae (CIP 104535T). We show here that these two species contain genes corresponding to all those of the M. smegmatis "GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in M. chelonae and M. abscessus. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (atf1 and atf2) in M. abscessus and M. chelonae has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (M. chelonae, 900 kb; M. abscessus, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins.ConclusionAlthough these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in M. smegmatis represents the ancestral form and that evolution has scattered various pieces throughout the genome in M. abscessus and M. chelonae.

SummaryThe cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap ( gap : GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence.

Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allothreoninyl residue and a 2-succinyl-3,4-di-O-CH 3 -rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.

Mycobacteria contain an outer membrane of unusually low permeability which contributes to their intrinsic resistance to many agents. It is assumed that small and hydrophilic antibiotics cross the outer membrane via porins, whereas hydrophobic antibiotics may diffuse through the membrane directly. A mutant of Mycobacterium smegmatis lacking the major porin MspA was used to examine the role of the porin pathway in antibiotic sensitivity. Deletion of the mspA gene caused high-level resistance of M. smegmatis to 256 g of ampicillin/ml by increasing the MIC 16-fold. The permeation of cephaloridine in the mspA mutant was reduced ninefold, and the resistance increased eightfold. This established a clear relationship between the activity and the outer membrane permeation of cephaloridine. Surprisingly, the MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the mspA mutant were increased 2-to 10-fold. This is in contrast to those for Escherichia coli, whose sensitivity to these agents was not affected by deletion of porin genes. Uptake of the very hydrophobic steroid chenodeoxycholate by the mspA mutant was retarded threefold, which supports the hypothesis that loss of MspA indirectly reduces the permeability by the lipid pathway. The multidrug resistance of the mspA mutant highlights the prominent role of outer membrane permeability for the sensitivity of M. smegmatis to antibiotics. An understanding of the pathways across the outer membrane is essential to the successful design of chemotherapeutic agents with activities against mycobacteria.The prevalence and spread of antibiotic resistance are increasingly serious problems that hamper the effective treatment of infectious diseases (26). The search for new antibiotics is mainly based on novel bacterial targets and high-throughput screening assays (10). However, many lead compounds discovered in vitro may fail because they do not reach their targets at sufficiently high concentrations in vivo (7). This is true in particular for gram-negative bacteria, which, in contrast to grampositive bacteria, are protected from the toxic actions of certain antibiotics, dyes, and detergents and host defense factors such as lysozyme by an additional outer membrane (OM) (49). The OM can be crossed by at least two general pathways: the hydrophobic (or lipid) pathway, which is characterized by the nature and the interactions of the membrane lipids, and the hydrophilic (or porin) pathway, whose properties are determined by water-filled channel proteins, the porins, which span the OM of gram-negative bacteria (49). It has been shown by the pioneering work of Nikaido and collaborators (21, 46) that Escherichia coli and Salmonella porins play a major role in the transport of ␤-lactam antibiotics. Subsequent studies showed that porin-deficient mutants of gram-negative bacteria were also more resistant to quinolones, tetracyclines, chloramphenicol, nalidixic acid, and trimethoprim (6,15,25,52). These data suggest that porins are involved in the transport...