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

Abstract

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. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is an extremely successful human pathogen. The pathogenesis of bacterium is associated with its ability to invade macrophages and to circumvent bactericidal functions of the host cell in order to survive within a protective niche. The cellular mechanisms are largely investigated but the bacterial factors are poorly known. The outermost layer of the mycobacterial cell envelope is particularly of interest because of its localization at the interface with macrophages. An interesting feature of this envelope is its high lipid content. One group of lipids, the phthiocerol dimycocerosates (DIM), has been studied intensively since being shown to promote Mtb virulence. We investigated the cellular and molecular mechanisms of DIM and demonstrated that DIM participate in the receptor-dependent phagocytosis of Mtb in human macrophages through a mechanism involving a reorganization of the plasma membrane following recognition of bacilli. This modification of the plasma membrane biophysical properties might help Mtb to create a protective niche by preventing acidification of its phagosome. Our results provide a first hint on the molecular mechanism of action of DIM, a key Mtb lipidic virulence factor.