The conical shape of DIM lipids promotes Mycobacterium tuberculosis infection of macrophages

Abstract

24Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen 25 Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into 26 macrophages, which occurs via phagocytosis, its molecular mechanism of action is 27 unknown. Here, we combined biophysical, cell biology, and modelling approaches to 28 reveal the molecular mechanism of DIM action on macrophage membranes leading to 29 the first step of Mtb infection. MALDI-TOF mass spectrometry showed that DIM 30 molecules are transferred from the Mtb envelope to macrophage membranes during 31 infection. Multi-scale molecular modeling and 31 P-NMR experiments revealed that DIM 32 adopts a conical shape in membranes and aggregate in the stalks formed between 33 two opposing lipid bilayers. Infection of macrophages pre-treated with lipids of various 34shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken 35 together, these results reveal how the molecular shape of a mycobacterial lipid can 36 modulate the biological function of macrophages. 37 38 we developed a multidisciplinary approach combining multiscale Molecular Dynamics 73 (MD) simulations, solid-state NMR and cell biology experiments. This revealed how 74 the molecular shape of DIM can affect macrophage membranes to promote 75 phagocytosis. 76 77 78 RESULTS 79 80 DIM are transferred to host cell membranes during macrophage infection 81First, we used MALDI-TOF mass spectrometry to assess whether DIM added 82 to host cells is incorporated into their membranes. Human macrophage (THP-1) cells 83 Figure 1: DIM are transferred from the bacterial envelope to macrophage membranes. (a) Structure of the DIM family of lipids, where m denotes the range of carbon atoms on the phthiocerol moiety, and n and p on the mycocerosate moieties. (b) MALDI-TOF mass spectra of purified DIM and of the membrane fraction of macrophages treated with DIM. (c) MALDI-TOF mass spectra of WT Mtb (HR37v) and of the membrane fraction of macrophages infected by H37Rv or by the H37RvDlppX mutant. M: low intensity peak corresponding to the detection of the matrix molecule in the DIM region of interest. The star symbol highlights the mass of the DIM molecule chosen for the modelling, with m=18, n=17, and p=4.

Lipid Specificity of the Fusion of Bacterial Extracellular Vesicles with the Host Membrane

“…LPS, the major component of MV owing to its relatively wider crosssection area and low head-to-tail aspect ratio could be a major contributor towards fluidization as it is known to uniformly adhere and incorporate in egg-PC, DOPG and DOPE containing lipid bilayer (37) all of which are present in the OLMM. This is in line with recent finding that Phthiocerol dimycocerosates (DIMs), a lipid component of Mycobacterium tuberculosis, are transferred to and accommodated in the host cell membrane due to its conical shape (38). The overall change in the fluidity of the host cell membrane would depend on the degree of interaction between bacterial MVs and the lipids specificity in the OLMM.…”

“…The changes in area per lipid molecule are coupled to the degree of interfacial adhesion/fusion of the interacting components (19,39). The observed increase in area per lipid molecule is likely due to the formation of inverted cubic structure/aggregates at the adhesion sites resulting in expulsion of some lipids (37,38,40), which, also explains the observed relatively higher negative values of ∆GExcess. Likewise, MV fusion to only DPPC membrane results in enhanced fluidization accompanied by increase in interfacial molecular dipoles (Fig.…”

Spreading of a mycobacterial cell surface lipid into host epithelial membranes promotes infectivity

“…These results were consistent with a recent report using cultured macrophages showing that M. tuberculosis PDIM occupies their cell membranes (Augenstreich et al, 2019).…”

“…Once spread, the potential for PDIM to disrupt membrane protein signaling is intriguing. Several biophysical properties of membranes are known to mediate membrane protein function (Los and Murata, 2004), and PDIM has been implicated in altering host membrane structure (Augenstreich et al, 2019). Furthermore, TLRs have been shown to aggregate into cholesterol rich lipid domains to facilitate downstream signaling events (Ruysschaert and Lonez, 2015).…”

“…There was no difference in DIM-488 spreading kinetics between wildtype and ∆RD1 M. marinum (Supplemental Figure 3C), suggesting that EsxA does not influence PDIM spreading. Besides playing a role in cytosolic escape, PDIM spreading into macrophage membranes has also been shown to promote phagocytosis of extracellular bacteria (Astarie-Dequeker et al, 2009;Augenstreich et al, 2019). To test this in vivo, we measured the rate of phagocytosis of wildtype or ∆mmpL7 M. marinum following HBV infection by measuring the number of discrete bacterial objects over time.…”