Basic understanding of mycobacterial pathways
Project: Mycobacterium ulcerans analgesia
ANR Grant AT2R Traak Bionanalgesics coordinated by Priscille BRODIN
Understanding pain, and relieving its symptoms, represent crucial challenges on scientific and medical grounds, with the available panoply of analgesics largely insufficient (Borsook et al., 2014). Notably to remedy this situation, we have witnessed recently a marked interest in the analgesic solutions elaborated by mother nature (Wood, 2013). The project builds on one such system we uncovered (Marion et al., 2014), with the toxin Mycolactone secreted by M. ulcerans antagonizing the pain of the lesions it inflicts.
This original achievement provides a unique opportunity to decipher, in full detail, the molecular and cellular pathways underlying pain control in this system involving AT2R receptors and TRAAK potassium channels. This approach will further allow to lay the ground for the elaboration of new rational strategies for the discovery of novel potent analgesic compounds. If the elucidation of the poorly understood AT2R-TRAAK system is the necessary condition for the projected discovery and development of analgesics, such understanding is in addition of interest, for its own sake, in the general background of GPCR family of receptors.
Indeed the triggering of the AT2R-TRAAK system by mycolactone relies on a complex pathway, and involves a series peculiarities (as compared to other GPCR systems), all of which can be put to advantage in the search for anti-pain molecules.
In terms of complexity, as compared to other recently uncovered models (concerning for example scorpions), the triggering of the signal with mycolactone involves a full cellular pathway, rather than a direct action on the ion channel involved. The elucidation of the various steps in such pathway then provides as many different potential handles to interfere with the process. In terms of peculiarities, in the general context of GPCR systems, there is no clear evidence in our case for a G-protein dependent signal transduction, and the triggering of the system does not appear to involve receptor internalization. Such a particularity, allowing to avoid desensitization, can then be of the highest interest in the context of potent analgesia. It will be important to determine how AT2R and TRAAK do interact, and notably if they form a complex. In addition, whereas our study has shown that mycolactone acts as an agonist for AT2R, previous studies had associated pain reduction with the action of antagonists for AT2R (possibly involving TRPV1 channels) (Danser and Anand, 2014). Such a possibility still widens the perspectives associated with AT2R as a privileged "anti-pain hub".
Furthermore, the extensive exploration of chemical variants of the toxin should lead to precious information for the rational engineering of new potent analgesics. It will be then also possible to extend the global view to various other natural systems, notably those involved in traditional medecines, in which the active compound appears to belong in various cases to the same lactone family than the toxin mycolactone (Kou et al., 2005).
Project: Mycobacterium tuberculosis pathogenesis
[M. tuberculosis /macrophages/control of acidification / Tirap-CISH-vATPase]
FRM and Univ. Lille funding
Stemming from our work (Queval et al., 2017), we will further characterize the complex formed by CISH and H+ V-ATPase at the vacuole through a higher spatial and temporal image-based approach. For the identification of other potential partners in the complex, we will perform a high throughput proteomic approach directly on M. tuberculosis containing phagosomes isolated from macrophages by immune-affinity separation. 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 M. tuberculosis containing vacuole will also be undertaken.
Recently, we could also show that the TLR adaptor Tirap (also called MAL) is required for STAT5 phosphorylation and CISH expression, which suggests that Tirap is critical for CISH-mediated bacterial control. Strikingly, the inhibition of bacterial replication is strikingly more pronounced in Tirap-/- compared to Cish-/- macrophages and mice suggesting a unique role of Tirap, which will be further elucidated.
Project: Mycobacterium tuberculosis pathogenesis
[M. tuberculosis /macrophages/ metabolomics reprogramming/ IRG1]
I-SITE ULNE ERC-Generator Grant
M. tuberculosis is engaging metabolic pathways of phagocytes to survive within host cells. In the mouse model of TB, exponential growth of M. tuberculosis occurs in the lung during the acute phase of infection, while the persistence phase is characterized by a metabolic shift initiated by changes in nutrient availability as substrates for glycolysis become limited. We will focus on the host metabolite itaconate, which has recently emerged as a regulator of macrophage function during inflammation and infection. Itaconate is induced in response to bacterial infections, LPS and proinflammatory stimuli that induce type I and type II interferon (IFN) signaling and is generated by the mitochondria-associated enzyme immune-responsive gene 1 (IRG1). Very recent findings in a mouse model of TB suggested that IRG1 deficiency is associated with severe pulmonary disease and mortality, with greater numbers of infected myeloid cells and higher production of inflammatory cytokines and chemokines (Nair et al., 2018). Our own work shows that IRG1 deficiency in M. tuberculosis-infected mice also leads to a dramatic reduction in T and B lymphocytes in the lung suggesting an additional role of IRG1 in controlling adaptive immunity during M. tuberculosis infection. Furthermore, in vitro experiments in macrophages showed that M. tuberculosis infection interferes with IRG1 expression and that IRG1 deficiency leads to increased M. tuberculosis replication rates. In addition, IRG1 is recruited to M. tuberculosis-containing vacuoles and affects one key characteristic of M. tuberculosis 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 M. tuberculosis infection, which influence mycobacterial growth and replication. We will further investigate the potential of IRG1-Itaconate axis in host-directed therapies against TB.
Therapeutic innovation in TB
Project: Small-molecules, antimicrobial peptides (AMP) and combination of against intracellular M. tuberculosis
[M. tuberculosis / macrophages / replication / AMP]
JPI AMR MTI4MDRTB
SATT Nord coordinated by Aurélie TASIEMSKI
The drug discovery efforts and focus on chemical biology in Lille includes medical chemistry, high content screening capacity, target identification, nanoparticles drug delivery systems and in vivo validation should definitely continue to position us as key player in the field of therapeutics against TB (Blondiaux et al., 2017). We will focus on drug candidates (either alone or in combination) having efficacy against M. tuberculosis replication within macrophages (also called antivirulence compounds or host-directed therapy). Our approaches will rely on i) the repurposing of commercially available drugs; and ii) the development of proprietary antimicrobial peptides (AMP) brought by Aurélie TASIEMSKI and François MASSOL joining the team in 2020.
They identified a new antibiotic peptide, alvinellacin, from Alvinella pompejana, the emblematic worm that inhabits the hottest part of deep sea hydrothermal chimneys of the East Pacific Rise (Papot et al., 2017; Tasiemski et al., 2014) This patented peptide (Aurélie Tasiemski et al., Patent number: 8652514 / Univ Lille) is the first active molecule against Mtb isolated from an extremophile animal and shows no homology to other molecules already described from terrestrial or marine organisms. The peptide folds into a double-stranded antiparallel β-sheet (β-hairpin motif) stabilized by two cysteine-bonds, which is unique to worms so far. Together with other molecules belonging to the same gene family and all issued from extremophile animals, their development against TB will be pursued using our efficient hit-to-lead optimisation pipeline. A. Tasiemski is invited to several oceanographic cruises giving access to extremophile organisms.
Project: Risk of resistance development of novel TB drug candidates
[M. tuberculosis / macrophages / resistance / antibiotics]
Theoretical models of biological evolution will be developed to predict the conditions of emergence of M. tuberculosis resistance against the proposed treatments, using the modelling frameworks of quantitative genetics and adaptive dynamics (Massol, 2013; Massol & Débarre, 2015). The prediction model will be confronted to the results of selection of resistant strains during long-term exposure to sub-lethal doses of drugs on the M. tuberculosis infected macrophages. Different models will be designed to account for possible genetic correlations between tolerance and resistance and will incorporate different ingredients which can potentially enhance the acquisition of resistance in bacteria such as i) the capacity for tolerance against antibiotics (i.e. survival through a decrease in intracellular bacterial growth), ii) the type of interaction linking the immune system to pathogens with respect to host resources, iii) source-sink dynamics between reservoirs of pathogen resistance acquisition and immunity-deprived patients.