Biology subjects

Blaise, M.

Publications and source records attributed to Blaise, M..

9 recordsLinked to original sources

bioRxiv2026

SARS-CoV-2 nucleocapsid protein engages with viral RNA and ERGIC lipids to drive viral core assembly

Severe acute respiratory syndrome coronavirus 2 assembles at the ER-Golgi intermediate compartment (ERGIC), yet the molecular basis of nucleocapsid (N) protein interactions with host membranes remains unclear. Using in vitro reconstituted lipid membranes and viral RNA- N complexes, we show that full-length N binds phosphatidylinositol (PI)- and phosphatidylserine (PS)-containing membranes and induces lipid clustering, an effect amplified by viral RNA and ERGIC-like membrane lipid composition. The isolated N-terminal domain lacks this activity, whereas the C-terminal domain retains membrane-associated multimerization. Although, lipid bilayers promote co-clustering of N and PI lipids, facilitating ribonucleoprotein (RNP) assembly, even on simple membranes. Importantly, ERGIC- mimicking membranes enhances this co-clustering further stabilizing RNPs of dimensions matching the viral core. In cells, viral RNA enhances N clustering without altering particle production. These findings reveal cooperative interactions between N, viral RNA, and ERGIC lipids as key drivers of lipid-dependent viral core formation, providing a mechanistic framework for the early steps of viral assembly.

biophysics↗

bioRxiv2026

Biochemical, structural, and functional characterization of the Nocardia asteroides dihydrofolate reductase: a primary target of anti-nocardiosis treatment

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs target respectively the dihydrofolate synthase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFRNad). We show that overexpression of DHFRNad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFRNad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFRNad bound to trimethoprim at high resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance. HighlightsFirst biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFRNad induces high-level trimethoprim resistance in N. asteroides. Crystal structure of DHFRNad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC50 data confirm strong DHFRNad inhibition by trimethoprim and methotrexate

microbiology↗

bioRxiv2025

NCY-I beta-lactamase activity correlates with antimicrobial susceptibility of a clinical strain of Nocardia cyriacigeorgica

Nocardiosis is an infectious disease caused by several Nocardia species, among which Nocardia cyriacigeorgica is one of the most frequently isolated species in the clinic. Albeit most isolates of this species are susceptible to standard treatment combining trimethoprim and sulfamethoxazole, resistance has been reported, necessitating alternative or combination therapies. {beta}-lactam antibiotics are of particular interest in this context. In this study, we aimed to address the {beta}-lactam susceptibility profile of a clinical strain of N. cyriacigeorgica and assessed whether it correlated with the enzymatic activity of purified {beta}-lactamase of the strain. We herein established that the strain is highly susceptible to imipenem and ceftriaxone, moderately sensitive to meropenem and resistant to amoxicillin. The resistance could be counteracted by {beta}-lactamase inhibitors from two distinct chemical classes: vaborbactam, and avibactam while clavulanate was less potent. We demonstrated that the {beta}-lactam susceptibility of the strain is in direct line with the enzymatic activity of purified NCY-I, a class A {beta}-lactamase. NCY-I was indeed only active with amoxicillin but displayed poor activity towards other classes of {beta}-lactams. The NCY-I activity could be inhibited in vitro by vaborbactam, clavulanate and avibactam. We consolidated these data by determining the high-resolution structure of NCY-I bound to avibactam. The structural analysis supported a conserved inhibitor binding site among other Nocardia class A {beta}-lactamases strongly suggesting a broad inhibition spectrum of avibactam across Nocardia species.

microbiology↗

bioRxiv2025

Role of the locus coeruleus noradrenergic system in susceptibility and resilience following early life stress in male and female mice

Child adversity (CA), encompassing emotional, physical, and sexual maltreatment or abuse, affects a substantial number of children worldwide. Moreover, it is the leading predictor of psychiatric disorders such as major depressive disorder (MDD), anxiety, and suicidal behavior. Despite the robust link between CA and psychopathology, individual outcomes vary significantly, with some children demonstrating resilience. Resilience is an adaptive and dynamic process, which mitigates the long-term effects of CA, suggesting potential protective mechanisms that remain underexplored. This study investigates behavioural susceptibility and resilience following early life stress (ELS) and examined whether these outcomes are associated with sex-dependent alteration of the locus coeruleus-norepinephrine (LC-NE) system, a critical modulator of stress, cognition, and emotion. Using a maternal deprivation model combined with limited nesting and bedding, we examined sex-dependent behavioral, physiological, and neurobiological markers associated with ELS outcomes in mice. Behavioral clustering revealed distinct phenotypes: resilient, anxious, and anhedonic-like with sex-specific differences in distribution. Early markers, including body weight and ultrasonic vocalization (USV) patterns, were linked to long-term susceptibility. Neuroanatomical analyses identified sex-specific LC-NE activation patterns associated with resilient and susceptible phenotype, particularly within the caudal-dorsal LC. These findings highlight sex-dependent behavioural trajectories of ELS that are associated with distinct LC-NE activation patterns, offering insights into potential interventions targeting resilience mechanisms in children exposed to CA.

neuroscience↗

bioRxiv2025

SNX-BAR proteins 5 and 6 are required for NCOA7-AS antiviral activity against influenza A virus

Interferon-induced antiviral proteins act on the first line of defence against viruses, including influenza A virus (IAV). Among those, the short isoform of Nuclear Receptor Coactivator 7 (NCOA7-AS) has been shown to inhibit IAV entry and more especially the fusion between endosomal and viral membranes. Ectopic expression of NCOA7-AS leads to the increased acidification of the endolysosomal pathway, putatively through NCOA7-AS interaction with the vacuolar ATPase (V-ATPase). However, the precise mechanism of action of NCOA7-AS is not fully understood. Here, we identified the cellular partners of NCOA7-AS by mass spectrometry, including the V-ATPase subunits known to interact with NCOA7-AS. A point mutation disrupting the interaction with the V-ATPase led to loss of antiviral activity against IAV, demonstrating that V-ATPase engagement is required for the antiviral phenotype. Moreover, sorting nexin (SNX) 1/2/5/6 were identified as novel partners of NCOA7-AS. These proteins are involved in retrograde transport of cellular cargoes from endosomes to the trans-Golgi network. We revealed that SNX5/6 interacted directly with NCOA7-AS and were essential for NCOA7-AS antiviral activity against IAV. Interestingly, crystal structures of NCOA7-AS/SNX5 complexes showed that the SNX5-interaction motif in NCOA7-AS was similar to those found in known cargoes of SNX5/6. In addition, we could pinpoint several critical residues that were important for binding and antiviral activity. Collectively, our study identifies novel essential partners for NCOA7-AS antiviral activity and the structural basis for their interaction.

microbiology↗

bioRxiv2024

Single-stranded DNA drives sigma subunit loading onto RNA polymerase to unlock initiation-competent conformations

Initiation of transcription requires the formation of the "open" promoter complex (RPo). For this, the {sigma} subunit of bacterial RNA polymerase (RNAP) binds to the non-template strand of the -10 element sequence of promoters and nucleates DNA unwinding. This is accompanied by a cascade of conformational changes on RNAP the mechanics of which remains elusive. Here, using single-molecule Forster resonance energy transfer and cryo-electron microscopy, we explored the conformational landscape of RNAP from the human pathogen Mycobacterium tuberculosis upon binding to a single-stranded DNA fragment that includes the -10 element sequence (-10 ssDNA). We found that like the transcription activator RbpA, -10 ssDNA induced {sigma} subunit loading onto the DNA/RNA channels of RNAP. This triggered RNAP clamp closure and unswiveling that are required for RPo formation and RNA synthesis initiation. Our results reveal a mechanism of ssDNA-guided RNAP maturation and identify the {sigma} subunit as a regulator of RNAP conformational dynamics

molecular biology↗

bioRxiv2023

Crystal structure of a Class A beta-lactamase from Nocardia cyriacigeorgica

Nocardia are gram-positive bacteria from the Actinobacteria phylum. Some Nocardia species can infect humans and are usually considered opportunist pathogens as they often infect immunocompromised patients. Albeit, their clinical incidence is low, many Nocardia species are nowadays considered emerging pathogens. Primary sites of infection of Nocardia are the skin or the lungs but dissemination to other body parts is very frequent. These disseminated infections are very difficult to treat and thus, tackled with multiple classes of antibiotics, on top of the traditional treatment targeting the folate pathway. {beta}-lactams are often included in the regimen but many Nocardia species present moderate or strong resistance to some of this drug class. Genomic, microbiological, and biochemical studies have reported the presence of class A {beta}-lactamases (ABL) in a handful of Nocardia species but no structural investigation of Nocardia {beta}-lactamases has been performed yet. In this study, we report the expression, purification, and preliminary biochemical characterization of the ABL from a Nocardia cyriacigeorgica (NCY-1) clinical strain. We describe, as well, the crystallization and the very high-resolution crystal structure of NCY-1. The protein sequence and structural analysis demonstrate that NCY-1 belongs to {beta}-lactamase of class A1 and attest to its very high conservation with ABL from other human pathogenic Nocardia. In addition, the presence of one molecule of citrate tightly bound in the catalytic site of the enzyme is described. This structure may provide a solid basis for future drug development to specifically target Nocardia spp. {beta}-lactamases. SynopsisThe crystal structure at high resolution of a class A {beta}-lactamase from a clinical strain of Nocardia cyriacigeorgica is reported.

biochemistry↗

bioRxiv2022

A tripartite complex HIV-1 Tat-cyclophilin A-capsid protein enables Tat encapsidation that is required for HIV-1 infectivity

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes but it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolylisomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated. Here we found that a Tat-CypA-CA tripartite complex assembles in HIV-1 infected cells. Biochemical and biophysical studies showed that high affinity interactions drive the assembly of this complex. Virions devoid of encapsidated Tat showed a 5-10 fold decrease in HIV-infectivity and, conversely, encapsidating Tat into {Delta}Tat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreases the efficiency of retrotranscription by [~]50% and transcription by 99%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust HIV-1 infection and viral production.

microbiology↗

bioRxiv2022

The N-terminal domain of MX1 proteins is essential for their antiviral activity against different families of RNA viruses

Myxovirus resistance protein 1 (MX1) and MX2, are homologous, dynamin-like large GTPases, induced upon interferon (IFN) exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus (IAV), by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with an amino-terminal GTPase domain and a carboxy-terminal stalk domain, connected by a Bundle Signalling Element (BSE). These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against IAV and Vesicular Stomatitis Virus (VSV), and for the ability to aggregate Orthobunyavirus nucleoproteins. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities, but does modify MX1 protein subcellular localisation. The discovery of this essential and highly conserved residue defines this region as key and may reveal insights as to the mechanism of action of MX1 proteins.

microbiology↗