Biology subjects

Saillant, V.

Publications and source records attributed to Saillant, V..

3 recordsLinked to original sources

bioRxiv2025

Identification and optimization of trans-species reactive TfR1-binding VHH as tools for drug delivery across the blood brain barrier

The blood-brain barrier (BBB) is a major obstacle for delivering therapeutic agents to the central nervous system (CNS), posing significant challenges for treating neurological disorders. Among current strategies to improve brain drug exposure, hijacking physiological pathways involved in receptor-mediated transcytosis (RMT) has emerged as a promising strategy. While targeting transferrin receptor 1 (TfR1) is widely explored, many TfR1-antibodies lack cross-species reactivity, limiting translational development. In the present study, we identified and characterized camelid-derived single-domain antibodies (VHHs) with robust cross-reactivity to rodent, rhesus monkey, and human TfR1. Epitope mapping of the VHH revealed a novel binding site at the interface of the TfR1 dimer. When fused to a human IgG1 Fc domain, these VHHs, as monomers or homodimers, were efficiently internalized by engineered CHO cells and brain endothelial cells expressing TfR1 from different species. Systemic administration of VHH-Fc constructs in mice demonstrated significantly improved brain uptake compared to irrelevant controls. Functional delivery was confirmed using neurotensin (NT)-induced hypothermia, and we established correlations between in vivo effects and TfR1 VHH binding properties determined by surface plasmon resonance. Notably, efficient BBB transcytosis was associated with intermediate affinity and rapid dissociation rates. Engineered variants maintained favorable cross-species binding, including similar affinities to human and non-human primate TfR1, facilitating translational studies. The cross-reactive anti-TfR1 VHHs we developed offer a versatile and modular platform for CNS drug delivery and hold promise as molecular shuttles for transporting therapeutic agents across the BBB. Our work establishes a robust foundation for developing next-generation brain-targeted biotherapeutics, including peptides, enzyme replacement therapies, antibody-based treatments for neurodegenerative diseases, and oligonucleotide delivery for CNS disorders, enabling seamless translation from preclinical to clinical applications.

neuroscience↗

bioRxiv2022

HssS activation by membrane heme defines a paradigm for 2-component system signaling in Staphylococcus aureus.

Strict management of intracellular heme pools, which are both toxic and beneficial, can be crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, heme is embedded in the membrane bilayer with its 2 protruding porphyrin propionates interacting with 2 conserved Arg94 and Arg163 that are located extracellularly. Mutagenesis of these arginines and of 2 highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic heme binding pocket abolished the ability of HssS to induce HrtBA synthesis. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation to induce HrtBA-mediated heme extrusion from the membrane. This "gatekeeper" mechanism could limit intracellular diffusion of exogenous heme in S. aureus, and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors. ImportanceIn the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity, Staphylococcus aureus expresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphorelay mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the TM helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen sensing mechanisms as a prerequisite to the discovery of inhibitors.

microbiology↗

bioRxiv2020

A novel Enterococcus faecalis heme transport regulator (FhtR) is a heme sensor in the gastrointestinal tract.

Enterococcus faecalis is a commensal Gram-positive pathogen found in the intestines of mammals, and is also a leading cause of severe infections occurring mainly among antibiotic-treated dysbiotic hospitalized patients. Like most intestinal bacteria, E. faecalis does not synthesize heme. Nevertheless, environmental heme can improve E. faecalis fitness by activating respiration metabolism and a catalase that limits hydrogen peroxide stress. Since free heme also generates redox toxicity, its intracellular levels need to be strictly controlled. Here, we describe a unique transcriptional regulator, FhtR, (Faecalis heme transport Regulator), which manages heme homeostasis by controlling an HrtBA-like efflux pump (named HrtBAEf). We show that FhtR, by managing intracellular heme concentration, regulates the functional expression of the heme dependent catalase A (KatA), thus participating in heme detoxification. The biochemical features of FhtR binding to DNA, and its interactions with heme that induce efflux, are characterized. The FhtR-HrtBAEf system is shown to be relevant in a mouse intestinal model. We further show that FhtR senses heme from blood and hemoglobin but also from crossfeeding by Escherichia coli. These findings bring to light the central role of FhtR heme sensing in response to heme fluctuations within the gastrointestinal tract, which allow this pathogen to limit heme toxicity while ensuring expression of an oxidative defense system. ImportanceEnterococcus faecalis, a normal, harmless colonizer of the human intestinal flora can cause severe infectious diseases in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote its resistance and its virulence. Here, we report a new mechanism used by E. faecalis to detect the concentration of heme, an essential but toxic metabolite that is present in the intestine. E. faecalis needs to scavenge this molecule to respire and fight stress generated by oxydants. Heme sensing triggers the synthesis of a heme efflux pump that balances the amount of heme inside the bacteria. With this mechanism, E. faecalis can use heme without suffering from its toxicity.

microbiology↗