Research - New entrants - 2023

The severe acute respiratory syndrome coronavirus (SARS-CoV/CoV-2) genome encodes 16 non-structural proteins (nsps), which coordinate cell remodeling, virus replication and participate in viral evasion. Notably, nsp3 contains a protein module termed Macro domain, which carries IFN antagonist activity that interferes with host innate immunity response. This domain is able to bind and hydrolyze ADP-ribose derivatives. This activity is correlated to viral escape and thus makes Macro domains a valuable therapeutic target. In the present paper, we report a SARS-CoV Macro domain structure in complex with a MOPS molecule. Based on our structural data, molecular docking was performed on a set of MOPS analogs in the ADP-ribose binding pocket. We present an ELISA-based assay to select hits based on the inhibition of recombinant SARS-CoV/CoV-2 Macro domain-ADP-ribose complex formation. Among the tested analogs, MOPSO and CAPSO are the more efficient in inhibiting ADP-ribose-binding. Structural analysis of these molecules in the ADP-ribose pocket reveals potential interactions with amino acid residues involved in the coordination of ADP-ribose. Overall, these findings suggest that MOPSO and CAPSO bear potential to be used as a scaffold for the design of Macro domain-specific inhibitors.

Brown algae are key components of coastal ecosystems worldwide, also providing significant environmental and economic value to society through the development of sustainable seaweed mariculture for food and biomass production. They also make a substantial contribution to primary production and carbon sequestration, burying several megatons of carbon from the atmosphere per year, comparable to that reported for terrestrial forests.

Due to their early diverging position in the eukaryotic tree, brown algae are an outstanding source of original biosynthetic pathways, most of which are still unknown so far. Among them, the biosynthetic pathways of storage sugars (i.e. mannitol and laminarin), which represent a substantial part of algal biomass, have been poorly described so far. Therefore, this project aimed to functionally decipher the storage carbohydrate biosynthetic pathways in brown algae.

Based on functional genetics and heterologous expression approaches, we have successfully started the functional characterization of enzymes involved in mannitol biosynthesis and transport. This project has laid a solid foundation for the development of this new research topic in the BIAM (sugar and lipid metabolism in brown macroalgae), with recent fundings from the CEA and the ANR.

This project aimed at deciphering the molecular mechanism of interspecies cell-cell communication in complex microbial communities using a synthetic bacterial consortium composed of two workhouses organism E. coli and N. vulgaris. These are ubiquitous and can be found in various environments (soil, gut microbiota, etc.). Previous studies pointed towards the involvement of Quorum Sensing (QS) through the secretion of AI2 signal in triggering collective behaviors (including cell-cell interactions, metabolic cross-feeding, etc.) between these two microorganisms. While AI2 type QS mechanism has been well characterized in E. coli, little is known on QS mechanisms in N. vulgaris. Using bioinformatic survey, we showed that N. vulgaris lacks the gene involved in the AI2 synthesis (luxS). Nevertheless, we have identified in the genome of N. vulgaris homologs of lsr genes potentially involved in signal transduction via the phosphorylation of AI2 inside the cell (DVU_3134 coding for LsrK-like) and a transcriptional repressor (DVU_3131 coding for LsrR-like). This suggested that even though N. vulgaris cannot produce AI2, it might sense the signal in the environment. Hence, to challenge the role of interspecies QS mechanism in the synthetic ecosystem, this proposal was articulated into 2 work packages. WP1: First, the role of the putative QS actors was validated in vitro, using a combination of enzymatic, gel shift, and trans-complementation assays. The results obtained here, clearly pointed towards a functional AI2 QS pathway in D. vulgaris involved in the regulation of its metabolism. WP2: Whether AI2 could trigger metabolic coupling between different members in complex communities remained to be challenged. For this purpose, the E. coli and N. vulgaris synthetic consortium was used as model of simplified community. First, bacterial cooperation between these two bacteria was validated. Then, we showed that the presence of N. vulgaris impact the AI2 signal accumulation in the co-culture supporting a role of AI2 in interspecies bacterial communication. The exact mechanism of AI2 QS is currently under investigation using mutant strains and RT-qPCR analysis.

Tuberculosis (TB) is still a major health issue worldwide. The standard anti-TB chemotherapy is composed of 4 antibiotics that require to be administered daily over at least 6 months. Within this combination, pyrazinamide (PZA) plays a key role by drastically reducing the duration of the treatment and limiting relapses. However, despite being used clinically for almost 50 years, the molecular mode of action (MoA) of PZA remains poorly characterized with conflicted models reported.

In this project, we aimed at challenging the current views regarding PZA MoA and solve this unique mystery.

By developing innovative CRISPRI approaches and combining it with standard antimicrobial susceptibility testing at various pH and fluorescence-based live recording of Mtb intrabacterial pH, we investigate what are the molecular factors driving PZA's ability to disrupt Mtb viability and intrabacterial pH homeostasis.