Recombinant protein expression in cells often leads to the formation of inclusion bodies (IBs), aggregates traditionally viewed as containing denatured, inactive proteins (Mayer & Buchner, 2003). However, recent studies have challenged this perception by revealing that IBs can harbor a significant portion of the cell's total protein content, with up to 95% purity. This dichotomy in IB composition distinguishes classical inclusion bodies (IBsC), characterized by compact, amorphous structures, from non-classical inclusion bodies (ncIBs), which exhibit a higher proportion of correctly folded proteins, facilitating their extraction and solubilization (Bucciantini et al., 2002); (Upadhyay et al., 2012).

This study focuses on delineating the parameters distinguishing classical IBs from ncIBs and optimizing production methods for proteins prone to aggregation. Through microscopic analysis, dynamic light scattering (DLS), and thermodynamic studies, we elucidated the distinct characteristics of these inclusion bodies. Our findings reveal that classical IBs form dense structures within bacteria, impacting cell division and localization, whereas ncIBs exhibit diffuse morphology in the cytoplasm. Moreover, while classical IBs necessitate denaturing agents for solubilization, hindering protein yields, ncIBs offer a promising alternative with their porous, less dense assembly, facilitating extraction using non-denaturing agents. By exploiting this distinction, we demonstrate the transformation of classical IB production into ncIB thereby enhancing yields of recalcitrant proteins.

Our results, supported by molecular, structural and biophysical techniques, underscore the potential of ncIBs as a platform for diverse protein production, offering avenues for enhancing yields and addressing challenges associated with hydrophobic and disordered proteins

 Biomass & Protein Engineering platform

Stagiaire: Yasmina BOUZARIOUH, étudiante en master Biologie structurale, génomique, parcours biochimie structurale

Bacteria have evolved sophisticated membrane channels, called secretins, specifically adapted for the assembly or secretion of bulky extracellular structures such as type 4 pili (T4P), needle complexes of type 3 secretion systems (T3SS) or large fully folded effectors in type 2 secretion systems (T2SS) (Review by Barbat et al., 2023b). We have recently shown that T2SS secretins must adopt different conformations to allow the passage of large effectors while maintaining the integrity of the bacterial envelope (Barbat et al., 2023a). In this project, we plan to develop and implement an ambitious in vivo experimental approach aimed not only at demonstrating the need for flexibility of the XcpD secretin during the secretion process, but also at identifying the partners that trigger these conformational changes. Specifically, we will further investigate the protein dynamics that exist between two adjacent secretin subunits by performing in-cell EPR experiments on spin-labelled proteins in their native environment (Review by Pierro et al., 2024) as well as in different genetic backgrounds lacking potential partners. With this approach, we hope to gain a better understanding of the different chronological steps that occur during the secretion process mediated by this singular nanomachine.

References:

Barbat, B., Douzi, B., Ball, G., Tribout, M., El Karkouri, K., Kellenberger, C., and Voulhoux, R. (2023a). Insights into dynamics and gating properties of T2SS secretins. Sci Adv 9, eadg6996.

Barbat, B., Douzi, B., and Voulhoux, R. (2023b). Structural lessons on bacterial secretins. Biochimie 205, 110-116.

Pierro, A., Bonucci, A., Magalon, A., Belle, V., and Mileo, E. (2024). Impact of Cellular Crowding on Protein Structural Dynamics Investigated by EPR Spectroscopy. Chem Rev 124, 9873-9898.

Redox enzymes are at the heart of a wide range of bioenergetic processes essential for all living organisms. They operate under mild conditions with high specificity, low overpotential and high turnover rates. Deciphering “how redox enzymes operate” is a prerequisite before harnessing their outstanding properties. Amongst a plethora of biophysical techniques, Surface Enhanced InfraRed Absorption (SEIRA) spectroelectrochemistry shines at probing redox enzymes in situ and in operando revealing information on secondary structure elements, orientation, catalytic mechanisms, redox chemistry and functionality.

However, despite this outstanding potential, SEIRA spectroelectrochemistry faces limitations mainly arising from the nature of the biological sample itself. In fact, it is nearly impossible to rely exclusively on intrinsic infrared markers of the protein to elucidate structural or environmental variations at a local level. Consequently, evaluating the immobilised redox enzyme orientation or observing protein dynamics is difficult as it is hampered by the global response of the protein amide bands. Similarly, monitoring catalysis at the redox active site is restricted to only a handful of enzymes.

The VIPER project aims at lifting off those limitations for : (1) improving SEIRA resolution and (2) broadening the range of investigable redox enzymes. The methodology will rely on the introduction of site-specific Vibrational Probes (VPs), into a model redox enzyme (Laccase). Vibrational probes are small molecules which vibrational mode is sensitive to its immediate environment in terms of electrostatics and H-bonding. Laccase catalytic activity couples the one electron oxidation of a wide range of organic and inorganic substrates to the four-electron reduction of molecular oxygen into water. We will decipher the orientation – activity relationships of laccase upon immobilisation onto electrodes. We will subsequently monitor its structural dynamics at a localised level and ultimately probe the various states of its catalytic cycle. This interdisciplinary project will pave the way for the spectroelectrochemical characterisation of poorly or yet uncharacterised redox enzymes.

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 administrated 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.

The bacterial secretory pathway (Sec) is the main process responsible for the localization of a large number of different essential proteins that need to be translocated across the cytoplasmic membrane into periplasm to be active. However, Sec substrate proteins are capable to assume misfolded conformations or aggregate in stress conditions, compromising the cell viability. Therefore, bacteria have evolved a complex network of various accessory proteins (molecular chaperones (MCs)) that cooperate to maintain in unfolded state the nascent proteins synthetized by the ribosome prior their translocation ( A. Tsirigotaki et al., Nat Rev Microbiol. 2017 15(1):21-36 ; 2) T. Cranford-smith et al. J. Biol. Chem., 295 (2020) 7516–7528).^. In 2020, a new MC related to Sec machinery, called YecA, has been identified in E.coli revealing a role in membrane biogenesis. This MC binds promiscuously various proteins while they are still nascent polypeptides and collaborates with other MC to maintain the translocation activity. The stress conditions required for its overexpression are currently not well understood, even if the iron availability seems to be one of the principal factors². Due to its recent discovery, YecA is yet poorly investigated and essential information are missing. In particular, the molecular mechanism for the recognition and the release of Sec-substrates, as well the interaction with other MCs and components of Sec still need to be investigated.

In this project, a structural and functional characterization of E.coli YecA will be carried out through the development of an innovative SDSL-EPR approach. Specifically, novel experiments based on SDSL-EPR spectroscopy will be carried out to decipher the interactions of YecA with other MCs and components of Sec pathway, focusing on the role of protein dynamics in the complexes formation. Combining EPR results with other biophysical techniques (ITC, DLC, CD, NMR and X-ray cryst.), the role of this MC in the E.coli Sec pathway and in the maintenance of bacterial proteome will be investigated.

As obligate intracellular parasites, viruses are dependent on their hosts for replication. This poses a unique challenge: how can the virus usurp cellular resources whilst avoiding detection by immune surveillance complexes? For viruses with positive sense RNA genomes (+ssRNA), genome replication is coupled to the remodelling of host membranes, yielding organelles that camouflage their RNA within the cell. All such organelles require a point of exchange with the cytosol, allowing for entry of cellular metabolites and exit of the nascently synthesized genome for translation and packaging into new virions. For several viruses including the alphavirus superfamily and coronaviruses, macro pores have been identified within the organelle membrane that may serve as a molecular gates.

This project proposed to structurally characterize these viral pores at high resolution with cryo electron microscopy. Pore complexes were studied following extraction with single particle methods, and a method to purify replication organelles for ex situ analysis of pores in their native membranes was also developed. Characterization of the pores will allow us to understand how they are integrated into viral replication, providing new avenues for therapeutics.

Cyanobacteria were the first organisms to evolve oxygenic photosynthesis, and now comprise a vast number of species, adapted to almost all the Earth’s environments. Some cyanobacteria can do photosynthesis using far-red light, a portion of the solar spectrum that is unused by most other oxygenic photoautotrophs because of its low energy content. These species are of high interest, because they could inspire biotechnological applications aimed at expanding the light usage of crop plants and microalgae into the far-red. Nonetheless, it is still unclear how the low energy content of far-red light affects the photosynthetic efficiency of these cyanobacteria, and which consequences this could have on the production of biomass.  In my previous postdoctoral research at IBPC in Paris, I adapted to cyanobacteria a spectroscopic method based on the ElectroChromic Shift (ECS) of photosynthetic pigments that is widely used in algae and plants to quantify the photosynthetic activity by measuring the trans-thylakoid electric field generated by electron transport. In the model species Synechocystis PCC6803 and Synechococcus elongatus PCC7942, I could show that ECS represents a new tool to measure the photosynthetic activity in vivo in cyanobacteria.

In the project funded by the IM2B, I adapted the ECS method to two far-red absorbing cyanobacteria species, Chroococcidiopsis thermalis PCC7203 and Acaryochloris marina. The funding IM2B was used to upgrade the spectroscopy instruments available in the P&E team at BIAM, and to build a dedicated incubator for the growth of the two far-red cyanobacteria species. Using the newly adapted ECS method, I could show that both Acaryochloris and Chroococcidiopsis use visible and far-red light with similar efficiency to sustain photosynthetic electron transport, despite the significant differences in their light absorption characteristics (https://doi.org/10.1016/j.bbabio.2024.149502 ). This is at variance with the majority of cyanobacteria, which cannot use the far-red portion of the light spectrum efficiently. ECS thus represents a new non-invasive tool to study the performance of naturally occurring far-red photosynthesis, potentially contributing to its future exploitation for sustainable biotechnological applications.