Evaluation of Amyloid Inhibitor Efficiency to Block Bacterial Survival.

Amyloid inhibitors, such as the green tea compound epigallocatechin gallate EGCG, apomorphine or curlicide, have antibacterial properties. Conversely, antibiotics such as tetracycline derivatives or rifampicin also affect eukaryotic amyloids formation and may be used to treat neurodegenerative diseases. This opens the possibility for existing drugs to be repurposed in view of new therapy, targeting amyloid-like proteins from eukaryotes to prokaryotes and conversely. Here we present how to evaluate the effect of these amyloid-forming inhibitors on bacterial amyloid self-assemblies in vitro and on bacterial survival. The different approaches possible are presented.

Nucleoside-Derived Low-Molecular-Weight Gelators as a Synthetic Microenvironment for 3D Cell Culture.

For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.

Apomorphine Targets the Pleiotropic Bacterial Regulator Hfq.

Hfq is a bacterial regulator with key roles in gene expression. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, thanks to its binding to small regulatory noncoding RNAs. This property is of primary importance for bacterial adaptation and survival in hosts. Small RNAs and Hfq are, for instance, involved in the response to antibiotics. Previous work has shown that the E. coli Hfq C-terminal region (Hfq-CTR) self-assembles into an amyloid structure. It was also demonstrated that the green tea compound EpiGallo Catechin Gallate (EGCG) binds to Hfq-CTR amyloid fibrils and remodels them into nonamyloid structures. Thus, compounds that target the amyloid region of Hfq may be used as antibacterial agents. Here, we show that another compound that inhibits amyloid formation, apomorphine, may also serve as a new antibacterial. Our results provide an alternative in order to repurpose apomorphine, commonly used in the treatment of Parkinson's disease, as an antibiotic to block bacterial adaptation to treat infections.

Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology.

Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system's components.

Self-Assembly of Nucleoside-Derived Low-Molecular-Weight Gelators: A Thermodynamics and Kinetics Study on Different Length Scales.

Biocompatible materials are of paramount importance in numerous fields. Unlike chemically bridge polymer-based hydrogels, low-molecular-weight gelators can form a reversible hydrogel as their structures rely on noncovalent interaction. Although many applications with this type of hydrogel can be envisioned, we still lack their understanding due to the complexity of their self-assembly process and the difficulty in predicting their behaviors (transition temperature, gelation kinetics, the impact of solvent, etc.). In this study, we extend the investigations of a series of nucleoside-derived gelators, which only differ by subtle chemical modifications. Using a multitechnique approach, we determined their thermodynamic and kinetic features on various scale (molecular to macro) in different conditions. Monitored at the supramolecular level by circular dichroism as well as macroscopic scales by rheology and turbidimetry, we found out that the sol-gel and gel-sol transitions are greatly dependent on the concentration and on the mechanisms that are probed. Self-assembly kinetics depends on hydrogel molecules and is modulated by temperature and solvent. This fundamental study provides insight on the impact of some parameters on the gelation process, such as concentration, cooling rate, and the nature of the solvent.

Interactions between DNA and the Hfq Amyloid-like Region Trigger a Viscoelastic Response.

Molecular transport of biomolecules plays a pivotal role in the machinery of life. Yet, this role is poorly understood due the lack of quantitative information. Here, the role and properties of the C-terminal region of Escherichia coli Hfq is reported, involved in controlling the flow of a DNA solution. A combination of experimental methodologies has been used to probe the interaction of Hfq with DNA and to measure the rheological properties of the complex. A physical gel with a temperature reversible elasticity modulus is formed due to the formation of noncovalent cross-links. The mechanical response of the complexes shows that they are inhomogeneous soft solids. Our experiments indicate that the Hfq C-terminal region could contribute to the genome's mechanical response. The reported viscoelasticity of the DNA-protein complex might have implications for cellular processes involving molecular transport of DNA or segments thereof.

Epigallocatechin Gallate Remodelling of Hfq Amyloid-Like Region Affects Escherichia coli Survival.

Hfq is a pleiotropic regulator that has key roles in the control of genetic expression. The protein noticeably regulates translation efficiency and RNA decay in Gram-negative bacteria, due to the Hfq-mediated interaction between small regulatory noncoding RNA and mRNA. This property is of primary importance for bacterial adaptation and virulence. We have previously shown that the Hfq E. coli protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure. In the present work, we demonstrate that epigallocatechin gallate (EGCG), a major green tea polyphenol compound, targets the Hfq amyloid region and can be used as a potential antibacterial agent. We analysed the effect of this compound on Hfq amyloid fibril stability and show that EGCG both disrupts Hfq-CTR fibrils and inhibits their formation. We show that, even if EGCG affects other bacterial amyloids, it also specifically targets Hfq-CTR in vivo. Our results provide an alternative approach for the utilisation of EGCG that may be used synergistically with conventional antibiotics to block bacterial adaptation and treat infections.

Revised role for Hfq bacterial regulator on DNA topology.

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNA. Besides these RNA-related functions, Hfq has also been described as one of the nucleoid associated proteins shaping the bacterial chromosome. Therefore, Hfq appears as a versatile nucleic acid-binding protein, which functions are probably even more numerous than those initially suggested. For instance, E. coli Hfq, and more precisely its C-terminal region (CTR), has been shown to induce DNA compaction into a condensed form. In this paper, we establish that DNA induces Hfq-CTR amyloidogenesis, resulting in a change of DNA local conformation. Furthermore, we clarify the effect of Hfq on DNA topology. Our results evidence that, even if the protein has a strong propensity to compact DNA thanks to its amyloid region, it does not affect overall DNA topology. We confirm however that hfq gene disruption influences plasmid supercoiling in vivo, indicating that the effect on DNA topology in former reports was indirect. Most likely, this effect is related to small regulatory sRNA-Hfq-based regulation of another protein that influences DNA supercoiling, possibly a nucleoid associated protein such as H-NS or Dps. Finally, we hypothesise that this indirect effect on DNA topology explains, at least partially, the previously reported effect of Hfq on plasmid replication efficiency.