The Polyketide Synthase Pks13 Catalyzes a Novel Mechanism of Lipid Transfer in Mycobacteria

SummaryMycolate-containing compounds constitute major strategic elements of the protective coat surrounding the tubercle bacillus. We have previously shown that FAAL32-Pks13 polyketide synthase catalyzes the condensation reaction, which produces α-alkyl β-ketoacids, direct precursors of mycolic acids. In contrast to the current biosynthesis model, we show here that Pks13 catalyzes itself the release of the neosynthesized products and demonstrate that this function is carried by its thioesterase-like domain. Most importantly, in agreement with the prediction of a trehalose-binding pocket in its catalytic site, this domain exhibits an acyltransferase activity and transfers Pks13’s products onto an acceptor molecule, mainly trehalose, leading to the formation of the trehalose monomycolate precursor. Thus, this work allows elucidation of the hinge step of the mycolate-containing compound biosynthesis pathway. Above all, it highlights a unique mechanism of transfer of polyketide synthase products in mycobacteria, which is distinct from the conventional intervention of the discrete polyketide-associated protein (Pap)-type acyltransferases

Lipoglycans Contribute to Innate Immune Detection of Mycobacteria

Innate immune recognition is based on the detection, by pattern recognition receptors (PRRs), of molecular structures that are unique to microorganisms. Lipoglycans are macromolecules specific to the cell envelope of mycobacteria and related genera. They have been described to be ligands, as purified molecules, of several PRRs, including the C-type lectins Mannose Receptor and DC-SIGN, as well as TLR2. However, whether they are really sensed by these receptors in the context of a bacterium infection remains unclear. To address this question, we used the model organism Mycobacterium smegmatis to generate mutants altered for the production of lipoglycans. Since their biosynthesis cannot be fully abrogated, we manipulated the biosynthesis pathway of GDP-Mannose to obtain some strains with either augmented (∼1.7 fold) or reduced (∼2 fold) production of lipoglycans. Interestingly, infection experiments demonstrated a direct correlation between the amount of lipoglycans in the bacterial cell envelope on one hand and the magnitude of innate immune signaling in TLR2 reporter cells, monocyte/macrophage THP-1 cell line and human dendritic cells, as revealed by NF-κB activation and IL-8 production, on the other hand. These data establish that lipoglycans are bona fide Microbe-Associated Molecular Patterns contributing to innate immune detection of mycobacteria, via TLR2 among other PRRs

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche

MmpL transporter-mediated export of cell-wall associated lipids and siderophores in mycobacteria

International audienc

Rôle de la 4'-phosphopantethéïnyl transférase PptT dans la multiplication et la persistance de Mycobacterium tuberculosis et mise en place d'un test d'activité enzymatique pour la recherche de nouveaux antituberculeux

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Etude des voies de biosynthèse des phtiocérol dimycocérosates, phénolglycolipides et des dérivés de l'acide para-hydroxybenzoïque (caractérisation du rôle des dérivés de l'acide para-hydroxybenzoïque dans la virulence de Mycobacterium tuberculosis)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

AUC is used as a start codon in Escherichia coli

International audienc

Disulfide Bridges Are Not Involved in Penicillin-Binding Protein 1b Dimerization in Escherichia coli

PBP1b can be found as a dimer in Escherichia coli. Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers