Pathogenic mycobacteria use fatty acids (FAs) released from host lipid droplets to fuel their central metabolism during infection. To import FAs, the bacteria are equipped with intricate transport machineries. However, the process of esterifying FAs with coenzyme A, an essential step for their further turnover, remains elusive.
Employing bioanalytical analysis such as thin layer chromatography and lipidomics, alongside advanced microscopy techniques like spinning disc imaging as well as structured illumination microscopy and serial-block face EM, we revealed that Mycobacterium marinum (Mmar) FA-CoA ligase (FACL) 6 is essential for FA esterification particular favoring C16 FAs. Deletion of FACL6 impedes FA uptake, reduces triacylglycerol levels and increases the sensitivity towards free FAs.
We postulate that unutilized free FAs interact with the membrane of the Mycobacterium-containing vacuole (MCV) rendering it susceptible to membrane damage. Indeed, when FACL6 is absent, markers for membrane repair (ALIX-GFP and GFP-VPS32) are recruited to the MCV more efficiently leading to an increase in phagosome escape. Consequently, the Mmar Dfacl6 mutant forms microcolonies in the host cytosol but is compromised by xenophagy and attenuated in Dictyostelium and in BV-2 cells. In immortalized BMDMs, FACL6 deletion triggers both plasma and phagosome membrane damage, resulting in increased pyroptosis.
Understanding the interplay between lipotoxicity, membrane damage and cytosolic translocation is vital to develop novel host-directed therapies aimed at disrupting the sequential stages of the infection cycle (such as phagosome escape and cell-to-cell spreading by pyroptosis) in favor of the host cell.