Biology subjects

Ayme, G.

Publications and source records attributed to Ayme, G..

5 recordsLinked to original sources

bioRxiv2025

Segmented filamentous bacteria undergo a structural transition at their adhesive tip during unicellular to filament development

Segmented filamentous bacteria (SFB) are intestinal commensals that promote immune system development and pathogen protection through intimate attachment to the ileal epithelium. Attachment occurs via the tip of unicellular teardrop-shaped SFB, called intracellular offsprings (IOs), before outgrowth into filaments. To characterize this critical stage of the SFB life-cycle, we imaged SFB using cryo-electron microscopy and tomography. IOs were surrounded by a repetitive surface (S)-layer that became replaced by disordered hair-like structures uniquely at the tip. Upon outgrowth into filaments, the S-layer was exchanged for a morphologically distinct repetitive hair- like layer. The bacterial structures and morphological transition were conserved across SFB from mouse and rat origin, while growth of mouse-SFB under non-attachment conditions in a heterologous host affected the SFB tip length and relative proportion of the tip stages. Moreover, the filament tip displayed surface exposure of the major Th17 antigen, a ubiquitous cell wall protein, underscoring the unique properties of the adhesive tip. This study identifies a novel IO- specific S-layer and reveals a conserved developmental transition of the SFB tip surface including the transient appearance of structures consistent in location and timing with being involved in host cell attachment.

microbiology↗

bioRxiv2024

Intranasal delivery of a broadly neutralizing single domain antibody targeting ACE2 protects against SARS-CoV-2 infection

SARS-CoV-2 accumulates mutations over time leading to the emergence of variants, which become largely resistant to existing vaccines and spike protein-targeted antiviral treatment. Therefore, there is a need for other therapies with broad efficiency. Here, we targeted the angiotensin-converting enzyme 2 (ACE2), the major entry receptor for SARS-CoV-2. We purified three single domain heavy chain antibodies (VHHs) after immunization of an alpaca with the ectodomain of ACE2. These VHHs bound ACE2 with nanomolar affinity and specifically detected membrane-anchored ACE2. Two of them (B07 and B09) neutralized by a competitive mechanism multiple SARS-CoV-2 isolates, including Omicron variants (XBB.1.16.1; EG.5.1.3; BA.2.86.1), without impacting the proteolytic activity of the enzyme. Fusion of B07 with conventional Fc domain markedly improved its binding and neutralizing efficacy. This dimeric Fc-conjugated B07 (B07-Fc) recognized specific residues of the N-terminal helix 1 of ACE2. When administrated prophylactically and intranasally, B07-Fc induced a strong dose-dependent protection of mice expressing human ACE2 (K18-hACE2) from SARS-CoV-2 Omicron. Hamsters were weakly protected due to low binding of B07-Fc to hamster ACE2. These single domain antibodies targeting hACE2 represent potential broad-spectrum therapeutic candidates against any emerging viruses using ACE2 as a receptor. These inhalable neutralizing single domain antibodies also represent a non-invasive approach against respiratory viral infection.

microbiology↗

bioRxiv2023

Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor

The 7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, against the extracellular domain of the human 7-nAChR were generated through alpaca immunization with cells expressing a human 7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the 7-nAChR but not to the other major nAChR subtypes, 4{beta}2 and 3{beta}4. E3 acts as a slowly associating positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not precluding the desensitization of the receptor. An E3-E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with -bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

neuroscience↗

bioRxiv2023

An original potentiating mechanism revealed by the cryoEM structures of the human α7 nicotinic receptor in complex with nanobodies

The human 7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest to design ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of 7 ligands, we recently generated two nanobodies named E3 and C4 acting as positive and silent allosteric modulators respectively. Here, we solved the cryo-EM structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an active or desensitized conformation in the absence of orthosteric agonist, and mutational analysis shows a key contribution of a N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation which opens new avenues for drug design.

neuroscience↗

bioRxiv2022

Development of nanobodies as theranostic agents against CMY-2-like class C β-lactamases

Soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against enzymes may behave as potent inhibitors. The immunization of alpacas with the CMY-2 {beta}-lactamase led to the isolation of three VHHs that specifically recognized and inhibited CMY-2. The structure of the complex VHH cAbCMY-2(254)/CMY-2 was determined by X-ray crystallography. We showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes in the catalytic site. The {beta}-lactamase inhibition was found to follow a mixed profile with a predominant non-competitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binder. Our study identified a binding site that can be targeted by a new class of {beta}-lactamases inhibitors designed with the help of a peptidomimetic approach. Furthermore, the use of mono or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enable the development of the first generation of ELISA test for the detection of CMY-2 produced by resistant bacteria. IMPORTANCEThe still increasing antimicrobial resistance in human clinic or veterinary medicine is a major threat for modern chemotherapy. Beside the major caution in the use of current antibiotics, it is important to develop new classes of antibiotics. This work was focused on {beta}-lactamases that are the enzymes involved in the hydrolysis of the major class of antibiotics, the {beta}-lactam compounds. We selected camelid antibodies that inhibit CMY-2, a class C {beta}-lactamase produced by bacteria isolated from the veterinary and human settings. We characterized the conformational epitope present in CMY-2 in order to create a new family of inhibitors based on the paratope of the antibody. Finally, we designed a primary version of a detection system based on an ELISA using VHH and polyclonal antibodies.

biochemistry↗