Functional aspects and physisopathological impact of intracytoplasmic lipid inclusions in mycobacteria – ILIome
Functional aspects and physiopathological impact of intracytoplasmic lipid inclusions in mycobacteria
Tuberculosis (TB) caused by the pathogenic bacterium Mycobacterium tuberculosis (M.tb), is the deadliest infectious disease worldwide. Treating TB takes a minimum of 6-9 months. The emergence of drug resistant strains amplifies the incidences of TB, mainly resulting in failure of treatment and death. There is thus an urgent need for novel therapeutic targets and approaches.
Target determination
Infection with M.tb leads to the formation of granulomas in the lung, where some of the infected macrophages accumulate lipids in lipid bodies (LB) giving the cells a foamy appearance. In such foamy macrophages (FM), M.tb is able to hydrolyze host lipids into fatty acids that are reused as lipid reserves within intracytoplasmic lipid inclusions (ILI), which will provide carbon source during its growth and propagation. These ILIs therefore play a major role in the entry into dormancy and in the reactivation phase of the bacillus.<br />However, very little is known about the formation and degradation of these ILI, in particular which enzymes are involved in their synthesis as well as hydrolysis, in both the host and the mycobacteria. The knowledge of the molecules, enzymes or proteins, involved in these processes during the infection would open the way to the discovery of new treatments against mycobacterial related disease.<br />Indeed, blocking mechanism of accumulation/consumption of lipid could allow to control the metabolism of lipids and consequently the growth and the entry in dormancy of these pathogenic mycobacteria. <br />In this context, we used the proximity labeling technology based on the activity of APEX2, an ascorbate peroxidase able to biotinylate under in vivo conditions its neighbors within a 10 nm radius in order to determine the whole proteome present on ILI at different time point. <br />Our project aims to i) identify and validate in M. abs under in vitro and ex vivo conditions, the enzymes present on ILIs; and ii) study the physiological role of selected proteins during active replication, latency and reactivation phases in infected macrophages and in the zebrafish model.
The proximity labbelling approach coupled with mass spectrometry will be used to biotynylate, enrich, isolate and further identify the enzymes/proteins present at ILI surface.
Two robust experimental models based on ILI formation and degradation, thus focusing on the mycobacterial lipid metabolism during the dormancy and reactivation processes, will also be used to decipher the mode of acquisition and consumption of lipids by mycobacteria.
We have prepared samples from M. abscessus, allowing to perform mass spectrometry experiments to identify the biotinylated protein not involved in ILI formation and corresponding to the cell background. Various control experiments have been used to validate our mass spectrometry analysis of streptavidin-enriched samples. After identifying the biotinylated proteins corresponding to the background noise, we constructed plasmids coding for APEX2 fused to the 3 different homologue genes: tgs1, Rv1683 and HBHA from M. abscessus(Task 2.2).
These constructs were made in plasmids allowing constitutive production under the control of hsp60 promoter (pMV261), inducible under the control of the acetamide promoter (pMyC) or under the control of their own promoter.
After checking the production of its various fusion proteins in all growing conditions, we were able, to determine by mass spectrometry, the ILI proteome during the ILI formation at 24 and 48 hrs by using the ILI model developed in the lab. At 24 h, 123 proteins, were identified and 143 at 48 hrs. Interestingly, 38 proteins were in common between both time points and all other proteins were different. Bioinformatic analyses help us to determine the putative function of each protein and their relationship with the bacterial metabolism. This list of genes allows us to start the deletion mutants to study specifically the role of each protein. We decided to first focus our study on all proteins present at both time points with unknown function. In order to confirm in living cells if all proteins identified colocalized with ILI, selected genes have been fused with to gfp. all constructions of recombinant plasmids are available and they will be tested soon in living bacteria during ILI formation. If the co-localization with ILI is confirmed, we will start deleting these genes in M. abscessus. To conclude the interaction between all partners is good and the program should be respected by everybody.
For the next part of the project, we will generate samples for study the proteome of ILI degradation using the in vitro model and the mass spectrometry analyses will allow to determine the proteome involved during this process. Both studies will decipher the enzymes/proteins involved in ILI formation and degradation.
In parallel, we plan to start the same experiments in infected cells, where the bacteria use the lipids from the host to form its ILI.
During the same time, Partner 2 will finish the deletion mutant useful to study the physiological role of each target.
While Partner 3 will do the proteomic analyses, Partner 1 will start experiments on BMDM for KO mice planned on Task 1. We should have all homozygote mice during the end of the summer.
Even if we lost time due to mainly administrative troubles, the program of this year should be respected. Moreover, during the second part of this project, 2 or 3 studies should be ready for publications.
1. Intrabacterial lipid inclusions in mycobacteria: unexpected key players in survival and pathogenesis?. Mallick I, Santucci P, Poncin I, Point V, Kremer L, Cavalier JF, Canaan S. FEMS Microbiol Rev. 2021 May 24:fuab029. doi: 10.1093/femsre/fuab029.
2. Intrabacterial Lipid Inclusions: overview of an amazing organelle. Tonia Dargham, Ivy Mallick, Dominique Raze, Laurent Kremer and Stéphane Canaan, Elsevier.com/books and Journal, doi.org/10.1016/B978-0-323-91948-7.00003-8 253
M. tuberculosis, the causative agent of tuberculosis (TB), stores triglycerides (TAG) in the form of Intracytoplasmic Lipid Inclusions (ILI) either within foamy macrophages or inside the granuloma caseum center. These lipid-overloaded bacilli can persist silently within the host and establish a latent infection. During TB reactivation, degradation of TAG contained in ILI occurs, promoting bacterial growth and spread of the infection. This accumulation and degradation phenomenon of intracellular lipids is also observed in most mycobacteria. Thus, the ILIome project aims to study functional aspects and physisopathological impact of intracytoplasmic lipid inclusions in mycobacteria to 1) determine the contribution of host enzymes involved in ILI formation and degradation; 2) identify and characterize the mycobacterial proteins physically associated with ILI and participating either in ILI formation or consumption; 3) investigate the role of identified proteins in the mycobacterial pathogenicity through the use of deletion mutants in cellular and animal infection models. By using original techniques, this innovative project will provide key insights into the life cycle of pathogenic mycobacteria, inspiring probably innovative therapeutic strategies in the fight against TB.
Project coordination
Stephane Canaan (Laboratoire d'ingénierie des systèmes macromoléculaires)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partnership
CNRS DR12 - LISM Laboratoire d'ingénierie des systèmes macromoléculaires
IRIM Institut de Recherche en Infectiologie de Montpellier
CRCM Centre de recherche en cancérologie de Marseille
Help of the ANR 556,938 euros
Beginning and duration of the scientific project:
- 36 Months
