Inhibiting Fatty Acid Activation to Induce Phagosome Escape and Enhance Mycobacteria Clearance

• Applicant: Dr. Caroline Barisch
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 446405030

Tuberculosis is a highly infectious disease caused by Mycobacterium tuberculosis. Using Dictyostelium discoideum as model for foamy macrophages, we found that vacuolar mycobacteria access host lipid droplets to use the released fatty acids (FAs) as energy and carbon source during infection. To import FAs from their environment, mycobacteria are equipped with sophisticated transport machineries. However, the mechanism by which FAs are esterified with coenzyme A (referred to here as “FA activation”), an essential step for their further turnover, remains elusive. Our findings suggest that M. marinum fatty acid-CoA ligase (FACL) 6 plays a crucial role in activating C16 FAs during infection. Deletion of FACL6 results in nutrient deficiency (reflected by the incapacity to synthesize neutral lipids), elevated membrane damage and an increase in phagosome escape, probably to get access to the nutrient-rich cytosol. As consequence, bacteria lacking FACL6 are significantly attenuated since they are detected and eliminated by the xenophagy pathway. In addition, the bacteria become sensitive to an accumulation of potentially free FAs when FACL6 is absent. Presumably, free FAs accumulate in the Mycobacterium-containing vacuole (MCV) when host FA-activating enzymes, so called (very) long chain fatty acyl-CoA synthetases ((V)LC-FACS), are deleted. These proteins typically localize on endosomes and the MCV and have the function to esterify incoming exogenous FAs with coenzyme A. Deletion of LC-FACS1 & LC-FACS2 not only enhances the efficiency of M. marinum to escape from the MCV but also further potentiates phagosome escape of the M. marinum Δfacl6 mutant (i.e. EXIT pathway 2: damage of the vacuolar compartment and escape to the cytosol). In summary, nutrient deprivation and/or the enrichment of harmful free FAs drives the bacteria into the cytosol in which they are detected by the xenophagy pathway. Understanding the potential link between nutrient availability, lipotoxicity 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) in favour of the host cell. This will pave the way for novel treatment approaches that enhance the ability of the immune system to fight off the infection. Consequently, in this project, we will exploit D. discoideum to identify host proteins involved in FA activation during infection (Objective 1). Using mycobacteria defective in FACL6 as a tool to detect the accumulation of potential toxic free FAs, we will monitor the impact of host FA activation on mycobacteria proliferation, metabolic activity and phagosome escape (Objective 2). Finally, with the help of functional FA probes and FA-containing liposomes, we will specifically manipulate FA fluxes (Objective 3) to ultimately attenuate M. marinum in D. discoideum and M. tuberculosis in macrophages, respectively.

• DFG Programme: Priority Programmes
• Subproject of: SPP 2225: Exit strategies of intracellular pathogens