Structure and functions of cell envelope compartments
Mycobacteria react positively to the Gram stain and their cell envelope has been considered for a long time as being composed merely of a cell wall and a plasma membrane. Our demonstration of the occurrence of an outer membrane, called mycomembrane (MM, Fig. 1), whose thickness is similar to that of Gram-negative bacteria, now enlightens some of the unique properties of mycobacteria, such as their very low permeability to nutrients and hydrophilic drugs. In addition, we have shown that the capsular layer surrounding pathogenic mycobacterial species, in direct contact with the host immune system, is composed mainly of α-glucan, proteins and selective lipids. Therefore, targeting this envelope represents a key step towards anti-TB therapy. This required, however, the mapping of its individual components and understanding its organization in order to determine their role(s) of in mycobacterial physiology and pathogenicity.
Mycolic acids (MAs), α-alkyl, β-hydroxylated very-long chain (up to C100) fatty acids, are the major and specific components of the mycomembrane, esterifying the cell wall arabinogalactan and forming the inner leaflet of this membrane. They are essential for the survival of mycobacteria and crucial for their physiology and fitness. Furthermore, they play a determinant role in the virulence and the persistence of pathogenic mycobacteria within infected organisms, strongly potentiating the host immune response. In this context, we established the roles of two enzymes, HadC and HadD, involved in the synthesis of MAs in both Mtu and Msm. The inactivation of the coding genes not only strongly affects the structures of MAs, and the cell envelope permeability but also the virulence of the mutant strains in the mouse model (collaboration with the team of R. Brosch, Institut Pasteur, Paris). In collaboration with the teams of C. Guilhot (IPBS) and R. Brosch (Institut Pasteur, Paris) we correlated the absence of lipooligosaccharides in members of the Mtu complex with increased virulence in cellular- and animal-infection models. Altogether, these results suggest that mycobacterial surface remodelling enhance virulence driven evolution from “generalist” mycobacteria towards “professional” pathogens of mammalian hosts.
Although many constituents of the mycobacterial envelope have been described, the question of their specific location is still to be elucidated. To address this question, we dissected the cell envelopes of two rapid-growing mycobacterial species, M. aurum and M. smegmatis (Msm). We developed a method to isolate MM-containing cell wall fractions, where we identified the presence of Antigen85 complex proteins, known to transfer mycoloyl residues onto trehalose and cell wall polysaccharide arabinogalactan, porins and putative transporters, along with phospholipids, Mycobacterium-specific glycolipids and lipoglycans. This distribution differs significantly from that deduced from the widely used extraction method of mycobacterial cells with detergents. We showed, in collaboration with the teams of O. Schiltz and A. Milon (IPBS), that the unprecedented post-translational modification in bacterial proteins by O-mycoloylation we discovered some years ago occurs in several polypeptides of the Corynebacteriales order that comprises the Mycobacterium genus. We demonstrated that this O-mycoloylation of protein is essential for addressing proteins to the MM of Corynebacterium and sufficient for their assembly into mycolic acid-containing lipid bilayers. This illustrates how different molecules can organize within the envelope.
Our expertise in structural determination allowed us to take the lead in detecting, characterizing and disclosing biological activities of many lipids and glycoconjugates of the mycobacterial envelope. These include the characterization of the phtiocol, a pigmented naphthoquinone produced by Mtu, as a ligand whose sensing by AhR regulates antimycobacterial defence, in collaboration with S.H.E. Kaufmann (Max Planck Institute for Infection, Berlin). We also established the structure of a unique lipotripeptide typifying M. avium subsp. paratuberculosis (Map) ovine strains that promotes a cell-mediated immune response, in collaboration with the team of F. Biet (INRA, Tours), and patented as a marker in specific diagnosis. We also characterized the structures of several glycoconjugates involved in the biosynthetic pathway and transport of trehalose polyphleates produced by non-tuberculous mycobacteria, e.g. Msm, in collaboration with the team of C. Guilhot (IPBS).