Sébastien Brûlé

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF-derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two-phase interlaboratory benchmark using a shared V _H H sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant-reported apparent melting temperatures ( T _m ) showed good within-instrument repeatability but moderate inter-instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data-acceptance criteria in addition to instrument-dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent T _m depended strongly on the fluorescence readout. For this V _H H, single-wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF-derived apparent unfolding enthalpies were more variable than apparent T _m . Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument-specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data-quality criteria, and well-characterised reference materials to monitor instrument-dependent offsets.

In the deep Pacific and Indian oceans, Alvinellid worms diversified about 100 million years ago to colonize a variety of hydrothermal vent environments. It has been suggested that the last common ancestor of this family was a thermophilic species. However, the evolutionary history of these worms is complex, with putative gene flow among ancestors. In this study, we investigated the early evolution of the family in relation to the diversification of thermal niches. We demonstrated that phylogenetic histories of alvinellid species greatly vary along chromosomes, possibly due to allele introgression between nascent ecotypes. We performed sequence reconstructions of ancestral cytosolic MDH and Cu/Zn SOD under the two most frequent phylogenies encountered along the genome. In silico simulations of folding stability for these two enzymes were highly correlated with their biophysical characterization (micro-calorimetry and differential scanning fluorimetry), and were used to predict the thermostability of thousands of alternatively reconstructed proteins. We further generalized the reconstruction at the proteome scale, taking the amino-acid usage bias as a proxy for folding stability in a new phylogenetic model accounting for amino-acid variations over time. Both approaches agree that the last common ancestors of Alvinellidae gained highly stable proteins, comparable to proteins of present-day thermotolerant species. Our computational protocol allowed us to validate this scenario under multiple phylogenetic hypotheses and different sequence reconstruction models. Considered together, Alvinellidae shed light on how metazoan species diversify in order to colonize different thermal niches, combining adaptive mutation and selection on genetic variants. Significance: Deep-sea hydrothermal vents are among the most extreme environments on Earth. They, however, represent oases of life for a restricted number of highly specialized species. As such, Alvinellid worms constitute an exceptional family, including highly thermotolerant animals such as the Pompeii worm. Despite their vent endemicity, Alvinellidae thrive under contrasting ecological conditions, particularly regarding temperature. Understanding how this family diversified to colonize broad thermal regimes is of major importance for dissecting the mechanisms by which species adapt in highly unstable environments. Combining experimental protein resurrection, proteome-wide predictions, and new phylogenetic models, we assess the reliability of ancestral sequence reconstructions that represent molecular thermometers to evaluate past environmental conditions.

Cette thèse de doctorat en Validation des Acquis de l’Expérience présente un parcours consacré à la biophysique intégrative appliquée notamment aux macromolécules impliquées dans les maladies émergentes et ré-émergentes. Elle décrit la mise en place d’un service de contrôle qualité des protéines, désormais prérequis pour l’accès à la plateforme. Mon parcours met également en évidence une évolution de mon rôle dans de nombreuses collaborations scientifiques. Je suis passé d’un travail centré sur l’hydrodynamique à un champ plus large incluant la stabilité thermique et la spectroscopie. En parallèle, j’ai contribué à des développements méthodologiques et participé à la formation d’étudiants et chercheurs, ce qui a permis de diffuser et d’ancrer ces pratiques dans la communauté. L’originalité de ce parcours repose sur l’articulation entre service, recherche et développement méthodologique. Ce cadre me positionne aujourd’hui comme un interlocuteur privilégié pour ces thématiques dans l’environnement d’une plateforme technologique de pointe.

In this paper, we describe Ciimarium marinum gen. et sp. nov. (Prochlorococcaceae, Synechococcales) based on 16S rRNA gene phylogenies, 16S-23S ITS secondary structures, ultrastructure (TEM), morphological, pigments and ecological data of “Synechococcus” sp. LEGE 11466. The strain was isolated from a sample collected at Leixões Harbour, Portugal (41°11’08.1”N 8°43’08.8”W) on September 26th, 2011, by scraping an epilithic biofilm under 10-13m depth, less than 1km offshore. Bayesian Inference and Maximum Likelihood phylogenies demonstrate that the Ciimarium sequence is placed in a monophyletic clade with strong phylogenetic support (BI=1, ML=100), within the Prochlorococcaceae cluster. The type strain LEGE 11466 is phylogenetically distant from other reference strains of Cyanobacteria and is grouped with Uncultured “Synechococcus” MLS1228cl3 , “Synechococcus” MLCB, “Cyanobium” ATX 6A2 ATX-6A2-C45 and “Synechococcus” MBIC10613.. The secondary structures of 16S-23S ITS corroborate with the separation of Ciimarium from all the Prochlorococcaceae genera. Also, they show that MLCB and ATX 6A2 ATX-6A2-C45 may be different species from the type LEGE 11466. The ultrastructure analysis shows parietal thylakoids, in agreement with the family description. Morphologically the genus is indistinguishable from other Prochlorococcaceae genera. The pigments analysis shows Zeaxanthin and β-carotene as the main pigments, and also a low concentration of phycocyanin. Finally, our phylogenies indicate that a revision of the Prochlorococcaceae family is necessary, as there are still many Synechococcus-like Cyanobacteria strains that should be described as new genera in the future.

The morphology, 16S rRNA gene phylogeny and 16S–23S rRNA gene ITS secondary structures of three strains of marine Cyanobacteria, isolated from inter- and subtidal environments from north Portugal were studied, resulting in the description of Zarconia navalis gen. nov., sp. nov. (Oscillatoriales incertae sedis), Romeriopsis navalis gen. nov., sp. nov. (Leptolyngbyaceae) and Romeriopsis marina sp. nov., named under the International Code of Nomenclature for algae, fungi, and plants. No diacritical morphological characters were found for the new genera and species. The 16S rRNA gene maximum-likelihood and Bayesian phylogenies supported that the genus Zarconia is a member of the Oscillatoriales, morphologically similar to the genera Microcoleus and Phormidium, but distant from them. The genus Romeriopsis is positioned within the Leptolyngbyaceae (Synechococcales) and is closely related to Alkalinema . The secondary structures of the D1-D1′, Box B, V2 and V3 helices corroborate the phylogenetic results. Furthermore, our study supports previous observations of polyphyletic Oscillatoriales families and reinforces the need for their taxonomic revision.

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.

Mycolactone is a diffusible lipid toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer disease. Altough bacterially derived mycolactone has been shown to traffic from cutaneous foci of infection to the bloodstream, the mechanisms underpinning its access to systemic circulation and import by host cells remain largely unknown. Using biophysical and cell-based approaches, we demonstrate that mycolactone specific association to serum albumin and lipoproteins is necessary for its solubilization and is a major mechanism to regulate its bioavailability. We also demonstrate that Scavenger Receptor (SR)-B1 contributes to the cellular uptake of mycolactone. Overall, we suggest a new mechanism of transport and cell entry, challenging the dogma that the toxin enters host cells via passive diffusion.

To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome’s transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)₂(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451).

Intrinsic viscosity is a key hydrodynamic parameter to understand molecular structure and hydration, as well as intramolecular interactions. Commercially available instruments measure intrinsic viscosity by recording the macromolecular mobility in a capillary. These instruments monitor Taylor dispersion using an absorbance or fluorescence detector. By design, these instruments behave like U-tube viscometers. To our knowledge, there are no studies to date showing that the Viscosizer TD instrument (Malvern-Panalytical) is able to measure the intrinsic viscosity of macromolecules. In this study, we then performed our assays on the Poly(ethylene oxide) polymer (PEO), used classically as a standard for viscometry measurements and on three model proteins: the bovine serum albumin (BSA), the bevacizumab monoclonal antibody, and the RTX Repeat Domain (RD) of the adenylate cyclase toxin of Bordetella pertussis (CyaA). The presence of P20 in the samples is critical to get reliable results. The data obtained with our in-house protocol show a strong correlation with intrinsic viscosity values obtained using conventional techniques. However, with respect to them, our measurements could be performed at relatively low concentrations, between 2 and 5 mg/ml, using only 7 µL per injection. Altogether, our results show that the Viscosizer TD instrument is able to measure intrinsic viscosities in a straightforward manner. This simple and innovative approach should give a new boost to intrinsic viscosity measurements and should reignite the interest of biophysicists, immunologists, structural biologists and other researchers for this key physicochemical parameter.

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454–484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.