Bacteria are commonly defined as unicellular organisms; however, they constantly exchange substances and information with their confrères and the environment, and can efficiently shelter themselves and achieve homeostasis by building multicellular collaborative macrocolonies called biofilms. Members of these sessile communities can undergo significant functional differentiation and are typically embedded in complex extracellular matrix that secures both mechanical protection and a medium for intercellular exchange. Importantly, the switch between sessile and motile life-styles in pathogenic bacteria can correlate directly with the development of chronic vs. acute infections, whereas extracted bacterial matrix components can find a variety of beneficial biotechnological applications. Exopolysaccharides (EPS) are a major biofilm matrix component and are typically produced by trans-envelope secretion nanomachines, many of which are controlled at multiple levels by the intracellular second messenger c-di-GMP. Here, we will consolidate our expertise in biofilm formation, cyclic dinucleotide signaling, bacterial secretion and integrative structural biology to decipher EPS secretion system assembly and function in several medically and industrially relevant species. We will use complementary recombinant and in situ structural biology approaches together with established genetic and imaging techniques to decipher the molecular events controlling EPS biogenesis from transcription initiation, interdependent protein folding and cooperative subunit interactions; through secretion system assembly, formation of supramolecular secretory nanoarrays and EPS modifications; to harnessing the biosynthetic processes for the engineering of novel anti-infectives or beneficial EPS superproducers. Over the last years we have spearheaded these studies by unprecedented mechanistic insights into several secretion systems and have demonstrated the feasibility of such an ambitious project.

In public health contexts, where results of conventional imaging methodologies are often disappointing, molecular multimodal imaging is within a realm of possibility. Various imaging techniques are informative for functional groups, molecular weights, or special recognition sites, but no individual technique provides optimal answers to all questions. Thus, combining information from two or more measurement platforms is highly attractive. Such an approach is required to elucidate the complex spatial distribution of biomolecules in tissues, opening the way for a qualitative and quantitative multi-omics overview, both targeted and unbiased, of lipids, proteins, peptides, antibiotics, nucleic acids as well as glycans. Herein, we propose to combine vibrational spectroscopy, namely Raman Scattering (RS) and Mass Spectrometry Imaging (MSI) in a single workflow. Strengths and weaknesses of these technologies make them highly complementary. Key objectives of MultiRaMaS are both methodological and biological. On the one hand, as an emerging and challenging strategy multimodal imaging combining RS and Matrix-Assisted Laser Desorption/Ionization (MALDI) MSI needs further improvements: 1) optimization of a stringent and common sample preparation including cryo-sectioning and accurate identification of anatomical structures, 2) optimization of settings for fast multi-omics screening by RS imaging, 3) optimisation of settings to access the high chemical specificities and structure details by MALDI-MSI, 4) quantification of biomolecules of interest by MALDI-MSI, and 5) data processing including visualization, quantification, co-registration as well as fusion of hyperspectral imaging data. On the other hand, this original and remarkable approach, not fully explored, will be applied throughout a single tissue section and methodological outputs will be applied for the first time to the characterization of tuberculosis (TB), an infectious disease which affects each year 10 million humans and causes 1.7 million deaths. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, establishes a durable lung infection in complex lesions where it is found intracellularly in various immune cell types and extracellularly in the central necrotic core of these lesions. Our current efforts are focused on increasing spatial resolution to visualize and identify the structures of molecules of interest at the cellular and subcellular level, i.e. lifting a scientific barrier. Of particular interest is the mapping and quantification of anti-TB drugs and biomarkers in the phagolysosome of macrophages where M. tuberculosis bacilli reside. Thus, the major biological objectives are: 1) to decipher the architecture and the microenvironment of tuberculous lesions, 2) to map, identify and quantify anti-TB drugs and biomarkers in the phagolysosome of macrophages, and finally 3) to evaluate the effect on Mtb of anti-TB drug concentration and duration of treatment. As an ultimate objective, all imaging data will be used to apply artificial intelligence and machine learning to facilitate the automation of the superimposition of imaging data onto digitized and fully annotated histological images. To meet the strategic objectives, MultiRaMaS is organized into 5 tasks. MALDI-MSI experiments shall rely on a highly performant combination of a source with high spatial resolution and high resolution mass spectrometer combining high accuracy and efficient MS/MS capability. This project gathers the complementary expertise of the project coordinator and his renowned collaborators to ensure the development of a multimodal imaging workflow to investigate tuberculosis. Complementary resources and expertise are assembled to ensure the feasibility of the proposed scientific program. This multidisciplinary and interdisciplinary project is original because of the technologies implemented which have rarely been combined, as well as by its applications to investigate tuberculosis.

Protein-protein interactions (PPIs) play crucial roles in many biological and pathological processes and represent potential targets for developing new therapeutic approaches. Most targeted PPIs require the development of inhibitors to prevent the association of the two protein partners and abolish the resulting chain of biological events. However, the converse approach remains largely unexplored for therapeutic targets, and would modulate PPIs via stabilizers that reinforce their interactions. An example system is the case of cyclosporin A (CsA) which stabilizes the formation of the immunosuppressant complex CyclophilineA/Calcineurin (CypA/Cn). However, the small molecule CsA, commonly used in the treatment against graft rejection, has significant side effects mainly because of its lack of specificity for the Cn protein. The search for new strategies for stabilizing the CypA/Cn complex is therefore desirable. Synthetic foldamers have undergone a rapid development in the last decade, driven by the hope that they could achieve functions that match or even go beyond those of biopolymers. Among these, aromatic amide foldamers stand out thanks to their exceptionally predictable, tunable and stable conformations in solution, relatively easy synthesis of secondary and tertiary-like objects as large as small proteins, and a high amenability to crystal growth and structural elucidation. These features all point to aromatic amide foldamers as potential scaffolds to bear proteinogenic side chains and thus allow for ligands that target large protein surface areas. An interesting property of 8-fluoro/chloro-quinoline based foldamers developed in our team, is their ability to form hybridized double helices. This feature will be exploited to build supramolecular complexes stabilizing protein heterodimers, and in particular CypA/Cn within the framework of this project. A main question is how to rationally arrange proteinogenic side chains on a foldamer to bind to a given protein? Structure-based design is only possible if structures are available, and as yet, atomic details of foldamer-protein complexes have been challenging and are therefore scarce. Key preliminary results support the new possibility to obtain detailed structural information about interactions at a foldamer-protein interface even in the absence of strong binding, provided a type of attachment links the two molecules. The approach we propose falls within the field of dynamic combinatorial chemistry, by screening a library of foldamers equipped with disulfide terminations on the CypA and CnB proteins (Cn subunit B) for which a thiol will be introduced on the surface by mutagenesis (Xaa ? Cys). MS analysis will reveal the complexes of better affinity and will be used to guide design of the corresponding foldamer sequences. Further structural analysis will then be performed with these favourable sequences, by crystallography and by NMR in solution, using labeled proteins. The data obtained will be used to optimize foldamer-protein interactions, in particular using modeling tools. The iterative enhancement of the interactions should allow the design of double-helical hybridized foldamers, capable of dimerizing the CypA and Cn proteins in a supramolecular complex, even in the absence of covalent bonds. The data resulting from the proposed investigation will provide an essential framework toward future foldamers that could be used to target immunosuppression via the above protein-protein interactions. However, numerous steps that fall out of the scope of the proposed research would have to be made before following a therapeutic objective (evaluation of toxicity, metabolism, bioavailability, and immunogenicity). We nevertheless anticipate a significant impact in the specific field of biomolecular recognition by foldamers, or in the more general area of understanding communication between synthetic and biological molecules.

Many biological processes rely on multi-functional molecular machines, such as the ribosome (protein synthesis), bacterial pili and needles (host-pathogen contact) or bacteriophage (viral infection). These machines are built by the assembly of multiple copies of protein subunits into large macromolecular structures, also called supramolecular assemblies. Understanding assembly, structure and functions of these machines is hampered by the challenging structural characterization task. Indeed, supramolecular assemblies are non-crystalline objects, unable to form diffracting-quality crystals amenable to X-ray crystallography. Furthermore, the use of solution Nuclear Magnetic Resonance (NMR) is restricted by the inherent insolubility of these assemblies. Solid-state NMR (SSNMR) requires neither cristallinity nor solubility and can provide atomic details about structure and dynamics of biological assemblies. Notably, the first atomic models of a prion in its fibrillar form (the prion domain of HET-s, see Wasmer et al., Science 2008) and a bacterial filament (the Type III Secretion System needle, see Loquet et al., Nature 2012) were conceived on the basis of SSNMR restraints. In the project SUPRAMOL, I propose to use SSNMR to solve the structures of two biological supramolecular assemblies: the Type II Secretion System (T2SS) pseudopilus and the inner tube of the tail of the bacteriophage Mu. The T2SS is a complex machinery found in Gram-negative bacteria, designed to transport effector proteins from the bacterial periplasm to the extracellular space. T2SSs contain a periplasmic filament, called the pseudopilus, that promotes the protein secretion through the outer membrane. This filament is built by multiple copies of a single polypeptide subunit, forming an insoluble and non-crystalline macromolecular assembly. I will use SSNMR to solve the high-resolution structure of the T2SS pseudopilus from Klebsiella oxytoca in its native assembled state and to determine its specific interactions with its substrate, the pullulanase. Likewise, the inner tail tube of the bacteriophage Mu forms an insoluble and non-crystalline objet assembled by a single subunit protein. In its filamentous state, the inner tail tube is a hollow filament through which the DNA is transported, in order to prepare the DNA delivery into the host bacterium. I propose to use SSNMR to study the structure of the inner tail tube of the phage Mu in its native filamentous state.

Large molecular assemblies are ubiquitous in living cells and play key roles in many biological processes. Indeed, multiple copies of protein subunits can organize into large macromolecular nanostructures, in shapes of fibrils, filaments, pores, capsids, etc. Understanding the mechanisms responsible for the molecular assembly of such systems is of primary interest in biology in order to gain insights into their crucial functions. Inspired by these remarkable architectures, supramolecular chemists and material scientists aim at designing synthetic self-assemblies with a broad spectrum of applications, ranging from regenerative medecine to drug delivery. The design of new nanostructures and the tuning of their functionalities require a detailed description and understanding of the weak interactions driving the molecular assembly process. For self-assembled nanostructures involved in cellular processes or engineered by supramolecular chemistry, the 3D structure determination is hampered by several technical challenges: (i) the assemblies usually lack crystalline order required to perform X-ray crystallography and (ii) the high molecular weight prohibits fast molecular tumbling, which restricts the use of solution Nuclear Magnetic Resonance (NMR). So far, hybrid approaches combining high-resolution structures of the isolated subunits and the molecular envelope obtained from electron microscopy have led to few low- to medium-resolution models of such assemblies. However, this approach lacks the experimental determination of the crucial subunit-subunit interfaces, which can lead to inaccuracies since the subunits can adopt different conformations in isolation compared to their relevant assembled states. We have recently proposed a new approach based on modern solid-state NMR techniques to solve atomic structures of complex self-assembled nanostructures, demonstrated by an atomic model of a bacterial filament (Loquet et al., Nature 2012). This breakthrough forms the genuine basis of the NanoSSNMR project. We now envision disentangling more complex supramolecular self-organizations, either involved in synthetic or cellular processes. The NanoSSNMR proposal will exploit state-of-the-art solid-state NMR methods and strategic isotope labeling and integrate hybrid approaches to elucidate the assembly mechanisms, revealing the atomic structures of two complex self-assembled nanostructures.