SensiGut: Programmable bacterial biosensors for gut metabolites monitoring. Increasing evidence describes how gut microbiota influences human physiology. The emerging picture suggests that the interplay between the human genome and the microbiome is continuously regulating metabolic pathways and host response, which can influence the pathogenesis of chronic inflammatory diseases. Inflammatory bowel disease (IBD) has recently emerged as a public health challenge worldwide. The imbalance of key microbiome-derived metabolites is implicated in the pathogenesis of IBD. Routine monitoring of these IBD metabolite signatures in patients' samples might improve their temporal mapping for predicting disease course. However, current metabolomics techniques are impractical and expensive for daily routine monitoring. Therefore, new methods are needed to support fast, field-deployable, and cost-effective monitoring of metabolites in clinical samples. Here I aim to develop programmable whole-cell biosensors detecting metabolic biomarkers of IBD in clinical samples. Living cells perform parallel processing of various environmental signals and self-replicate, and are thus an attractive option to engineer affordable sensing devices. Here, I will develop an optimized bacterial biosensor chassis for operation in fecal samples and deliver a collection of reliable biosensors for IBD metabolite signatures. By applying my expertise: in synthetic biology, metabolic engineering, logic devices, and multicellular systems, I will deliver biosensors performing multiplexing logic for multiple and expanded metabolite detection in fecal samples. Their suitability to perform metabolic profiling will be evaluated in a clinical context using human samples from an established biobank in collaboration with Celine Deraison at IRSD, Toulouse, and clinicians at CHU Montpellier. The biosensor’s reliability will be tested by comparing the endogenous metabolites detected with metabolic analysis. This work will provide a solid tool for large-scale and daily metabolite monitoring in human samples, supporting new strategies for disease flares prevention and treatment. Ultimately, these sensors could be used by the patients themselves. These biosensors could support a wide number of epidemiological studies, such as in different populations, and ethnic groups for which differences in microbiota composition have been described. This strategy will have a valuable impact on IBD disease monitoring as well as on synthetic biology, microbiome research, epidemiology, and health-focused fields in the social sciences. These biosensors could be then re-applied to other pathologies, providing new, field-deployable metabolite monitoring and contributing to a deeper understanding of the complex chemical cross-talk between the gut microbiota and humans, relevant for many diseases. In the future, these biosensors will support the engineering of therapeutic bacteria designed to sense and respond to these metabolites and reduce inflammation in situ.

Molecular Dynamics (MD) simulations are an extremely valuable tool for complementing experimental techniques in the characterization of biomolecular structure and dynamics. In recent years, the combination of advanced simulation techniques with the increase of computing power has remarkably enhanced the predictive capabilities of MD simulations based on atomistic models and nowadays it is possible to accurately describe the relevant dynamics of proteins of small and medium size. Despite of these impressive improvements, the contribution of MD simulations to our understanding of protein behavior in the cell is still limited since proteins mostly perform their functions as 'biomolecular machines' by forming transient or stable interactions with other biomolecules. At this juncture, a viable strategy to tackle this formidable but urgent challenge is to devise multi-scale computational approaches which combine advanced simulation schemes with both atomistic and coarse-grained description. The goal of this project is to develop an integrated computational protocol to provide a realistic and detailed picture of protein functioning in physiological conditions. This project will focus on the characterization of three challenging yet biologically-important systems: i) Hsp70 molecular chaperones, ii) contractile tail-like machines, iii) Disordered C-terminal regions of GPCRs. These applications will provide the opportunity to combine atomistic simulations, strategies of advanced sampling and experimental data provided by collaborators for pushing the boundaries of biomolecular modeling

There is indeed an urgent need to develop treatments for COVID-19. COVID-19 pandemic is due to the SARS-CoV-2 coronavirus that infects human cells through binding of its spike S protein to cell receptors including the ACE2 protein. The entry of virus into the cell after binding to its receptor is still not clearly deciphered. It is expected that preventing SARS-CoV-2 cell entry and intracellular trafficking may prevent or revert the progression of COVID-19. The goal of this project is to identify drugs preventing or decreasing SARS-CoV-2 cell entry and trafficking. To shorten clinical applications, drugs already approved for human usage will be screened in view of repurposing towards COVID-19 treatment. The ubiquitously expressed mechanoenzyme dynamin (DNM2) is the key regulator of endocytosis and membrane trafficking as it mediates the fission of membrane to promote vesicle release from the plasma membrane and from other organelles. SARS-CoV-2 enter into cell through both dynamin-dependent endocytosis and direct membrane fusion, then traffic and mature inside the cell partly in vesicles. DNM2 inhibition is thus a promising target and was shown to prevent cell entry of a plethora of viruses including coronoviruses. We previously identified FDA-approved drugs that inhibit DNM2 GTPase activity in vitro, highlighting candidate drugs that will now be tested for their effect on SARS-CoV-2 cell entry. We also validated Spike protein-typed MLV pseudovirus bearing the common G614 variant and expressing GFP or luciferase as a surogate to assess SARS-CoV-2 cell entry and trafficking. To assess the efficacy of selected FDA-approved drugs to impair SARS-CoV-2 cell entry the project includes several workpackages : - WP1. SARS-CoV-2 pseudovirus production expressing GFP or luciferase. - WP2. In cellulo drug medium throughput screening for reducing SARS-CoV-2 pseudovirus entry, using BGM and Vero E6 cells that are permissive to SARS-CoV-2 infection. Cell toxicity and inhibition efficacy (IC50) will be determined. - WP3. Mechanism of inhibition, and dependence on dynamin. - WP4. Validation in human cells and determination of IC50. This work should lead to the validation of one or several FDA-approved drugs for the inhibition of SARS-CoV-2 cell entry in a cellular assay. Indeed, drugs have simple chemical structures that can have several biomolecule targets. Drug repurposing is advantageous as their clinical effects, potential toxicity and production are already mastered. Depending on the drug, massive amount of compound may be already available for use. Drugs that could decrease SARS-CoV-2 cell entry should reduce the unfavorable course and potentially the contagiousness of COVID-19. They could be used in infected individuals to better prevent the development of severe forms, and potentially as a prophylaxis on a short term period. Clinical trials for validation in Human and therapeutic usage could be envisaged by the end of this project (9 mo).

Almost any eukaryotic (non-bacterial) cell grows a cilium or flagellum - at some point in its life. These slender cell appendages serve multiple sensory and signaling functions, and, in addition, can be motile. Motile cilia and flagella play a key role in a number of fundamental life processes of cells and organisms: as examples, their regular bending waves propel sperm and pathogens, including trypanosomes (the pathogen of sleeping sickness). Inside our bodies, thousands of mechano-sensitive flagella on the surface of epithelial tissues sense blood flow or pump fluids, such as mucus in our airways and cerebrospinal fluid in our brain. During embryonic development, the chiral beat of cilia determine where left and right will be, by generating symmetry-breaking flows in a specific structure called the organizer. In all these examples, not only does the beat of cilia and flagella exert active forces on the surrounding fluid and sets it in motion but, conversely, external fluid flows exert hydrodynamic forces on beating cilia and flagella and change their beat. This wave form compliance renders the flagellum an active force sensor. Thus, cilia and flagella combine sensory and motility function in one. The interaction between complex fluid flows and actively beating cilia and flagella poses fundamental questions at the interface of physics and biology, with relevance for biological function: First, the mechano-responses of flagella play an important role for the mechano-navigation of flagellated swimmers, i.e. their ability to follow boundary surfaces as guidance cue or to actively swim up-stream in external currents, a process termed rheotaxis. In fact, this mechano-navigation has been attributed an important role in guiding sperm cells within the narrow oviduct on their sojourn to the egg, yet remains insufficiently understood. Next, collections of cilia on epithelial surfaces can show collective dynamics, such as phase-locking to a common frequency despite active noise, and meta-chronal waves (similar to a Mexican wave in a soccer stadium) that facilitate efficient fluid transport. Finally, mechano-sensing is found also in non-motile cilia, where elastic deflections of the cilium are detected by dedicated molecular force sensors (TRP-channels) to signal functional read-outs of fluid flow, e.g. during heart morphogenesis or kidney homeostasis. To date, we do not know in any quantitative terms how the flagellar beat responds to mechanical forces. This is partly due to the previous lack of high-precision imaging, but also the lack of a theoretical framework that can account for the many degrees of freedom of flagellar shape dynamics. Previously, we started to tackle this challenge and developed such a framework that integrates state-of-the-art data analysis of flagellar shape dynamics and realistic hydrodynamic simulations of the three-dimensional Stokes equation, to provide a concise description of the nonlinear dynamics of flagellar oscillations that singles out a small number of key degrees of freedom. Only such a reductionist approach makes it possible to run simulations fully parameterized by experiment. We propose an extension of this theoretical framework to concisely characterize active mechano-responses of beating cilia and flagella (using dimensionality reduction and limit cycle reconstruction to relate shape changes and hydrodynamic forces under different load scenarios). This will bring us into a prime position to address the nonlinear feedback loops between flagellar dynamics and external fluid flows, enabling us to generate mechanistic insight into how flagellar mechano-navigation works, as well as into the collective dynamics of many cilia and flagella. Thereby, using theory and established collaborations with experimental partners, we will generate fundamental insight into a ubiquitous model system for the role of mechanics for motility and development that was highly conserved during the course of evolution.