Research activity

Axis 1. Signalling at the Host-Mycobacterium interface for Mycolactone

The central aim of this axis is the development of new potent analgesics, building on the detailed elucidation of the AT2R-Traak model. To reach this aim the project involving 6 teams in France (Marsollier, Lebon, Sandoz, Boustié, Yeramian and Brodin) deploys a full-spectrum approach, from detailed molecular and cellular characterizations, to extensive exploratory screenings of natural compounds and in vivo pain assessments. The project is characterized by several promising strengths, as well as notable safeguards relative to the risks inherent to such approaches:
1) The effect of mycolactone appears to be very long lasting, thus comparing very favourably to compounds such as morphine, with the short duration of action being recognized as a severe shortcoming.
2) The system under study appears to be significantly more complex than other bioinspirational models for analgesia (for example scorpion toxins acting directly on ion channels), making the study more difficult, but offering from the applications perspective many different handles to interfere with the system.
3) The scientific approach adopted provides a sound safeguard relative to the medical objectives of the project, as the expected informations concerning the poorly characterized AT2R GPCR receptor and the Traak ionic channel will be valuable for their own sakes, notably in view of the increasing appreciation of the physiological importance of these components.

Axis 2. Impact of Host and Pathogen Heterogeneity on Phagosomal Rupture at the Host-Mycobacterium interface for Mycobacterium tuberculosis

Once embedded in the lung alveoli, Mtb is phagocytosed by lung cells, which are critical in the establishment of host immune response during infection. Once internalized by the host cell, Mtb has the strong capacity to replicate and persist. This feature relies on its ability to i) block phagosomal acidification, ii) exploit the cell death pathway and iii) induce phagosomal rupture in order to gain access to the cytosol. The cytosolic access of Mtb, which is so far restricted to virulent strains, has major consequences on the innate immunity that determine adaptive immune responses.

Among the currently known mycobacterial factors involved in the phagosomal rupture, the Type VII Secretion System (T7SS) ESX-1 secretion and the phthiocerol dimycocerosates of Mtb act PDIM play a primordial role (Augenstreich et al. 2017). However, the host factors involved in the ESX-1 mediated phagosomal rupture have not been characterized so far.

We showed that Mtb infection induced the TLR adaptor TIR-containing adaptor protein (Tirap) expression, which prevented phagosomal acidification and rupture (Belhaouane et al 2023). We further demonstrated that the Tirap-mediated anti-tuberculosis effect occurs through TLR2/STAT5/GM-CSF axis. We further demonstrated that Tirap controlled the expression of the Cytokine-inducible SH2 containing protein (CISH) and that both Tirap and CISH are enriched in Mtb-containing phagosomes. Subsequently, CISH targets the V-ATPase for ubiquitination, thus promoting its degradation (Queval et al 2017). Eventually, Tirap and Cish knock-out bone marrow derived macrophages are more resistant to Mtb replication relative to the wild type ones. We will now further characterize the complex formed by Tirap/CISH and H+ V-ATPase at the ruptured vacuole through a higher spatial and temporal in cellulo cryo-electron microscopy-based approach (cryo-CLEM) in collaboration with Jost Enninga (Institut Pasteur, Paris). Cryo-CLEM is an extension of Correlative Light Electron Microscopy allowing to integrate functional information on the involved host and bacterial factors in a dynamic fashion (Swistak et al 2021). Therefore, Mtb cytosolic access in comparison to other phagosome-rupturing pathogens can be studied at an unprecedented precision using this technique.

On the contrary, we also identified the mitochondria-associated enzyme immune-responsive gene 1 (IRG1) as host factor-dampening Mtb replication (Machelart et al. biorxiv). Similarly, to Tirap and CISH, IRG1 is recruited to Mtb-containing vacuoles. However, it additionally affects one key characteristic of Mtb infection, the accumulation and recruitment of lipid droplets to these vacuoles. These findings suggest that IRG1 is an important enzyme involved in the regulation of host metabolic responses to Mtb infection, which influence mycobacterial growth and replication alongside to a previous report (Nair et al 2018).

For the identification of other potential partners in the complex, we will perform a high throughput proteomic approach directly on Mtb containing phagosomes isolated from macrophages. We will further characterize the role of ubiquitination on the H+ V-ATPase. A more global approach aiming at deciphering the dual role of the host ubiquitinome at the Mtb containing vacuole will also be undertaken. Alternatively, we will question the role of Mtb phagosomal survival and the different processes involved with regard to Mtb growth ability (replicating/non-replicating) by using patient isolated persistent variants (in collaboration with O. DUMITRESCU and C. GENESTET). The goal of this axis is to describe the molecular processes involved in the interaction between Mtb and the phagosome, which lead to different outcomes depending on variations in both the host and the pathogen.