Research projects on mycobacteria

RESEARCH PROJECTS

Our projects detailed below are focussed on the study of various mycobacteria: M. smegmatis, M. bovis BCG, M. abscessus, M. marinum, M. ulcerans, M. canettii and M. tuberculosis.

Role of Intacytoplasmic Lipid Inclusion (ILI) and study of proteins involved in their formation as well as degradation

To better understand the physiological role of lipolytic enzymes in several processes like growth, survival in macrophages, ability to accumulate or degrade lipids from the host as well as persistence in mouse lungs, lipolytic enzymes genes are deleted or over-expressed and the corresponding mutant strains are used for in vivo studies (macrophage, zebrafish, mouse). Regulation of these specific genes during the infection and reactivation processes are also studied with our in vitro and ex vivo foamy macrophages model using microarrays and RNAseq techniques.

Dissecting the membrane lipid binding properties and lipase activity of Mycobacterium tuberculosis LipY domains, from FEBS J. (2019) 286:3164-3181. Delineating the physiological roles of the PE and catalytic domains of LipY in lipid consumption in mycobacteria-infected foamy macrophages, from Infect. Immun. (2018) 86:e00394-18. Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and mycobacterial hypervirulence in zebrafish, from Scientific Reports (2019) 9:8667.

Among the lipolytic enzymes, four of them: LipY (lipase), Rv0183 (monoglyceride lipase), Rv1984 (lipase) and Rv3452 (phospholipase), have proven to be important diagnostic tools. Immunoserological tests had shown that these four antigens can effectively discriminate patients with active TB from latent infected or BCG vaccinated individuals, with high specificity and sensitivity, suggesting that these proteins are produced in sufficient amount to induce an immune response in the infected patient.

Vaccination tools based on immunogenicity of lipolytic enzymes

LipC (Rv0220) is an immunogenic cell surface esterase of M. tuberculosis, from Infect. Immun. (2012) 80:243-253. M. tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active TB, from PLoS ONE (2011) 6:e25078.

Based on these interesting results, these enzymes could be the starting point for new vaccines against TB, but very little data are currently available on how they induce the immune system and about their ability to implement an effective protection against M. tb. Even though the use of an antigenic protein as one tool to achieve a good protection against the spread of the bacillus can represent quite a challenge, the use of recombinant strains overproducing such highly antigenic proteins, or the use of modified extracellular vesicles carrying these proteins could be an original and powerful way to induce a stable immune response able to protect patients.

In that context, we intent to induce protection against TB by vaccinating mice either with BCG strains overexpressing the aforementioned selected lipolytic enzymes, or with purified exosomes containing these enzymes.

Search for novel anti-tuberculous molecules

This part of our research is focused on drug discovery development to obtain lead compounds against TB. Among several compounds tested against M. tuberculosis, we have discovered two new families of molecules, named Cyclipostins and Cyclophostin (CyC) analogs and Oxadiazolone (OX) derivatives, able to inhibit lipolytic enzymes and exhibiting promising anti-tuberculous effect without any cytotoxic effects towards host cells.

These inhibitors have been used in an activity-based protein profiling approach to monitor mycobacterial infection in order to address the following issues: i) identify and validate (both in vitro and in vivo) the mycobacterial enzymes impacted by our inhibitors that lead to the clearance of the bacteria during M. tuberculosis extracellular growth and in infected macrophages; ii) investigate the effects of our compounds on resistant M. tuberculosis strains; and iii) use them as probes to decipher the physiological role of mycobacterial lipolytic enzymes during the mycobacterial phase of active replication, latency and reactivation in infected macrophages.

Cyclipostins and Cyclophostin analogs are promising compounds in the fight against TB, from Scientific Reports (2017) 7:11751; they inhibit the antigen 85C from M. tuberculosis both in vitro and in vivo, from J. Biol. Chem. (2018) 293:2755–2769; as well as TesA, a major thioesterase required for outer-envelope lipid biosynthesis in M. tuberculosis, from J. Mol. Biol. (2018) 430:5120-5136.

Such knowledge will provide major information on the susceptibility and the general spread of TB that would lead to the development of new therapeutic strategies for elimination of actively growing and/or latent bacilli from infected individuals, and hopefully against resistant TB strains.