All Abstracts

Mycobacteria predominantly rely on host-derived lipids as carbon and energy sources during infection, but the dynamics of lipid acquisition during intracellular growth remains to be explored. While residing inside cells, mycobacteria can access various lipid sources depending on their location inside the MCV (mycobacteria-containing vacuole) or in the cytosol. In this study, we aim to elucidate those dynamics using Dictyostelium discoideum/M. marinum as an infection model system, offering a well-established platform to investigate the interplay between intracellular pathogens and cell-autonomous defense mechanisms. We first showed that the genetic knock-out of mycobacterial systems involved in the import (Mce transporters) and utilization (Facl6 and LipY for incorporation, Icl1 for detoxification) of lipids led to significant intracellular growth defects in D. discoideum, while their initial capacity to infect cells was not altered, indicating a limited ability to replicate. Lipid profiling by thin-layer chromatography highlighted an unbalanced metabolism for some mutants, notably the ones affected in lipid incorporation, with a defect in the production of neutral lipids and a slight increase in cell-wall associated lipids such as PDIM/PGL. Investigations by immunofluorescence microscopy revealed an altered intracellular location, with some being affected in their ability to properly establish their MCV. We then investigated lipid transfer from the host to the bacterium by following lipid droplet (LDs) dynamics. LDs accumulate in the vicinity of MCV early during the infection and are eventually translocated inside the MCV where they provide a source of nutrients for M. marinum. By high-content microscopy, we showed that LD clustering close to the bacteria was significantly reduced when M. marinum failed to damage their MCV (using a strain mutated in its region of difference 1 (ΔRD1), encoding the ESX-1 secretion system), while the total cellular LD content remains similar. On the contrary, when bacteria were able to escape earlier to the cytosol (in absence of autophagy repair and restriction), a larger LD accumulation was observed. By using a fluorescent fatty acid analog, we confirmed the reduced lipid transfer from the host to M. marinum ΔRD1, underlining that the ability of M. marinum to induce damage to its compartment is somehow correlated to lipid acquisition. Overall, these results suggest that the use of host-derived lipids by mycobacteria might represent a crucial step early in manipulating the phagocytic process leading to MCV genesis. Further investigations are ongoing to understand the precise mechanism of LD translocation inside the MCV.