Biology subjects

Levesque, S.

Publications and source records attributed to Levesque, S..

11 recordsLinked to original sources

bioRxiv2026

Insights Into The Early Diversification of blaNDM-1 Genetic Contexts Following its Emergence in Acinetobacter species and Spread to Enterobacterales

Since its emergence, the New Delhi metallo-{beta}-lactamase (NDM) has become a major global antimicrobial resistance threat, enabling bacterial hosts to resist most {beta}-lactam antibiotics, including carbapenems, and spreading widely across bacterial species and geographic regions. Believed to have originated in Acinetobacter spp., blaNDM subsequently spread to Enterobacterales through mobile genetic elements, despite barriers to lateral gene transfer between these phylogenetically distant taxa. To investigate the genetic diversity and mechanisms underlying this interspecies transfer and subsequent global expansion, we sequenced and characterized 13 blaNDM-1-positive Gram-negative bacterial isolates collected in India in 2007, among the earliest known isolates harboring blaNDM-1. Clonal diversity was observed among both Escherichia coli and Klebsiella pneumoniae isolates. Comprehensive genomic analysis revealed that blaNDM-1 was consistently embedded within a conserved multidrug resistance region, often associated with mobile genetic elements involved in lateral gene transfer. Notably, the insertion sequence ISAba14 was identified in 6 of the 13 sequences, suggesting a potential role in the early dissemination of blaNDM-1 among diverse bacterial hosts. These findings contribute to our understanding of the evolutionary origins and early genetic context of blaNDM-1, highlighting the importance of continued genomic surveillance and functional studies to better understand the persistence and dissemination of carbapenem resistance.

molecular biology↗

bioRxiv2025

Autophagy disruption primes CAR-T cell metabolism for sustained rejection of ovarian tumors

T-cell based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy face substantial hurdles when confronting solid tumors such as ovarian cancer, where metabolic constraints in the tumor microenvironment limit T cell infiltration and function. In particular, T cells exposed to nutrient deprivation and hypoxia upregulate autophagy, a lysosomal degradation pathway that negatively regulates effector responses. Here, we used CRISPR-Cas9 to target a folate receptor alpha (FR) CAR expression cassette into the locus of the essential autophagy gene ATG5, thereby generating autophagy-deficient CAR-T cells in a single editing step. Targeted metabolite profiling revealed that deletion of ATG5 induced widespread metabolic reprogramming characterized by increased glucose and amino acid uptake. Functionally, ATG5-knockout CAR-T cells maintained high cytolytic activity when assayed in patient-derived ascites in vitro, and exhibited superior and long-lasting tumor control against ovarian tumors in vivo. Taken together, our results suggest that deletion of ATG5 metabolically primes CAR-T cells for enhanced cytotoxicity in immune-suppressive conditions, thereby improving the therapeutic potential of FR CAR-T cells for ovarian cancer immunotherapy.

immunology↗

bioRxiv2025

In cellulo DNA assembly for targeted genomic integration and rearrangement in human cells

Although therapeutic genome editing holds great potential to remedy diverse inherited and acquired disorders, targeted installation of medium to large sized genomic modifications in therapeutically relevant cells remains challenging. We have developed an approach that permits DNA sequence assembly and integration in human cells leveraging CRISPR-targeted dual flap synthesis. This method, named prime assembly, allows for RNA-programmable site-specific integration of single- or double-stranded DNA fragments. Unlike homology-directed repair, prime assembly was similarly active in dividing and non-dividing cells. We applied prime assembly to perform targeted exon recoding, transgene integration, and megabase-scale rearrangements, including at therapeutically relevant loci in primary human cells. Prime assembly expands the capabilities of genome engineering by enabling the targeted integration of medium to large sized DNA sequences without relying on double-stranded DNA donors, nuclease-driven double strand breaks, or cell cycle progression.

molecular biology↗

bioRxiv2023

Nucleotide metabolism constrains prime editing in hematopoietic stem and progenitor cells

Therapeutic prime editing of hematopoietic stem and progenitor cells (HSPCs) holds great potential to remedy blood disorders. Since quiescent cells have low nucleotide levels and resist retroviral infection, we hypothesized that nucleotide metabolism could limit reverse transcription mediated prime editing in HSPCs. We demonstrate that deoxynucleoside supplementation and Vpx-mediated degradation of SAMHD1 improve prime editing efficiency in HSPCs, especially when coupled with editing approaches that evade mismatch repair.

genetics↗

bioRxiv2023

Emergence of Acinetobacter baumannii International Clone 10 predominantly found in the Middle East

Acinetobacter baumannii is a globally distributed human pathogen. Infections caused by carbapenem-resistant isolates of A. baumannii (CRAB) are of great concern, as treatment options are very limited. Despite having among the highest rates reported worldwide, there exists limited genomic data from CRAB strains isolated in the Middle East. Here we report epidemiological, phenotypic, and genome sequencing data (short reads and long reads) on a set of 60 A. baumannii isolates belonging to Sequence Type ST158 (Pasteur MLST scheme). They represent a novel international clone (IC), designated IC10, with limited geographic spread beyond the Middle East. Specific antibiotic-resistance genes associated with this clone were identified and data on the plasmid content associated with this lineage are presented.

microbiology↗

bioRxiv2023

MTOR as a selectable genomic harbor for CRISPR-engineered CAR-T cell therapy

Chimeric antigen receptors (CARs) reprogram T cells to recognize and target cancer cells. Despite remarkable responses observed with CAR-T cell therapy in patients with hematological malignancies, CAR-T cell engineering still relies mostly on randomly integrating vectors, limiting the possibilities of fine-tuning T cell function. Here, we designed a CRISPR-based marker-free selection strategy to simultaneously target a therapeutic transgene and a gain-of-function mutation to the MTOR locus to enrich cells resistant to rapamycin, a clinically used immunosuppressant. We readily engineered rapamycin-resistant (RapaR) CAR-T cells by targeting CAR expression cassettes to the MTOR locus. Using in vitro cytotoxicity assays, and a humanized mouse model of acute lymphoblastic leukemia, we show that RapaR-CAR-T cells can efficiently target CD19+ leukemia cells in presence of immunosuppressing doses of rapamycin. Furthermore, our strategy allows multiplexed targeting of rapamycin-regulated immunoreceptors complexes (DARICs) to the MTOR and TRAC loci to pharmacologically control CAR-T cells activity. We foresee that our approach could both facilitate the enrichment of CRISPR-engineered CAR-T cells ex vivo and in vivo while improving tumor eradication.

cancer biology↗

bioRxiv2023

Earliest observation of the tetracycline destructase tet(X3)

Tigecycline is an antibiotic of last resort for infections with carbapenem-resistant Acinetobacter baumannii. Plasmids harboring variants of the tetracycline destructase tet(X) promote rising tigecycline resistance rates. We report the earliest observation of tet(X3) in a clinical strain predating tigecyclines commercialization, suggesting selective pressures other than tigecycline contributed to its emergence.

microbiology↗

bioRxiv2023

SARS-CoV-2 spike antigen-specific B cell and antibody responses in pre-vaccination period COVID-19 convalescent males and females with or without post-covid condition

BackgroundFollowing SARS-CoV-2 infection a significant proportion of convalescent individuals develop the post-COVID condition (PCC) that is characterized by wide spectrum of symptoms encompassing various organs. Even though the underlying pathophysiology of PCC is not known, detection of viral transcripts and antigens in tissues other than lungs raise the possibility that PCC may be a consequence of aberrant immune response to the viral antigens. To test this hypothesis, we evaluated B cell and antibody responses to the SARS-CoV-2 antigens in PCC patients who experienced mild COVID-19 disease during the pre-vaccination period of COVID-19 pandemic. MethodsThe study subjects included unvaccinated male and female subjects who developed PCC or not (No-PCC) after clearing RT-PCR confirmed mild COVID-19 infection. SARS-CoV-2 D614G and omicron RBD specific B cell subsets in peripheral circulation were assessed by flow cytometry. IgG, IgG3 and IgA antibody titers toward RBD, spike and nucleocapsid antigens in the plasma were evaluated by ELISA. ResultsThe frequency of the B cells specific to D614G-RBD were comparable in convalescent groups with and without PCC in both males and females. Notably, in females with PCC, the anti-D614G RBD specific double negative (IgD-CD27-) B cells showed significant correlation with the number of symptoms at acute of infection. Anti-spike antibody responses were also higher at 3 months post-infection in females who developed PCC, but not in the male PCC group. On the other hand, the male PCC group also showed consistently high anti-RBD IgG responses compared to all other groups. ConclusionsThe antibody responses to the spike protein, but not the RBD-specific B cell responses diverge between convalescent males and females, and those who develop PCC or not. Our findings suggest that sex-related factors may also be involved in the development of PCC via modulating antibody responses to the SARS-CoV-2 antigens. Short SummaryPost-COVID Condition (PCC) is lingering illness that afflicts a significant proportion of COVID-19 patients from three months after clearing SARS-CoV-2 infection. Therapy for PCC is only palliative and the underlying disease mechanisms are unclear. The wide spectrum of PCC symptoms that can affect different organs and the detection of viral components in tissues distant from lungs raise the possibility that PCC may be associated with aberrant immune response due to presence of viral antigens. Therefore, we studied B cell and antibody responses to the spike and nucleoprotein antigens in PCC patients who cleared mild SARS-CoV-2 infection during the pre-vaccination COVID-19 pandemic period. We observed divergent patterns of immune reactivity to the spike protein in PCC males and females at different times post-infection, suggesting that the immune responses in PCC may also be influenced by sex-related factors.

immunology↗

bioRxiv2022

Molecular epidemiology of carbapenemase-producing Acinetobacter spp. from Israel, 2001-2006: earliest report of blaNDM predating the oldest known blaNDM-positive strains

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAb) is a WHO priority 1 critical pathogen. Despite early emergence of elevated CRAb rates in Israel, limited molecular data from this location are available. We searched for carbapenemases among 198 clinical Acinetobacter spp. from Israel between 2001 and 2006. MethodsStrains from 3 archives underwent whole-genome sequencing (Illumina NovaSeq on all, MinION on a subset) and computational analyses: assembly (Unicycler), annotation (prokka), identification (Kraken, rpoB similarity), search for carbapenemases (ResFinder, BLDB curation). FindingsA. baumannii (Ab) represented 179 (90{middle dot}4%) Acinetobacter spp. Eighty-four Ab (46{middle dot}9%) carried a carbapenemase: 38 (45{middle dot}2%) blaOXA-72 (blaOXA-24-like); 28 (33{middle dot}3%) blaOXA-23-like (20 blaOXA-23 and 8 blaOXA-225); 18 (21{middle dot}5%) blaOXA-58 (16 from 2001-2). Carbapenemase rates increased yearly from 2002 (32%) to 2006 (67%). Eight species of non-baumannii Acinetobacter (NbA) accounted for 19 isolates (9{middle dot}6%). Two of three A. junii contained blaOXA-58, one of which, Ajun-H1-3, isolated in January 2004, also possessed blaNDM-1. The pNDM-Ajun-H1-3 plasmid matched numerous NDM-positive plasmids reported from 2005 onwards in Acinetobacter spp. as well as Enterobacterales. InterpretationWe assessed carbapenemase diversity among Acinetobacter spp. in Israel from 2001-2006. Findings in Ab predate observations elsewhere: rapidly rising carbapenemase rates, driven by blaOXA-23-like and blaOXA-24-like genes replacing blaOXA-58. Among NbA, an A. junii isolated in 2004 carried blaNDM-1, making it the earliest NDM-positive isolate reported to date, preceding those from 2005 in India. Further research into blaNDMs emergence is warranted, in order to shed light on the evolution and spread of this and other antibiotic-resistance genes. FundingCentre de recherche Charles-Le Moyne; Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Universite de Sherbrooke; Fonds de recherche du Quebec - Sante; New Frontiers in Research Fund Grant NFRFE-2019-00444; CIFAR-Azrieli Global Scholars Program.

microbiology↗

bioRxiv2021

Marker-free coselection for successive rounds of prime editing in human cells

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we designed a robust coselection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineered highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process revealed that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific coselection strategies.

molecular biology↗

bioRxiv2019

Pangenome analysis reveals genetic isolation in Campylobacter hyointestinalis subspecies adapted to different mammalian hosts

Campylobacter hyointestinalis is an emerging pathogen currently divided in two subspecies: C. hyointestinalis subsp. lawsonii which is restricted to pigs, and C. hyointestinalis subsp. hyointestinalis which can be found in a much wider range of mammalian hosts. Despite C. hyointestinalis has been reported as an emerging pathogen, its evolutionary and host-associated diversification patterns are still vastly unexplored. For this reason, we whole-genome sequenced 13 C. hyointestinalis subsp. hyointestinalis strains and performed a comprehensive comparative analysis including publicly available genomes of C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii to gain insight into the genomic variation of these differentially-adapted subspecies. Both subspecies are distinct phylogenetic lineages which present a barrier to homologous recombination, suggesting genetic isolation. This is further supported by accessory gene patterns that recapitulate the core genome phylogeny. Additionally, C. hyointestinalis subsp. hyointestinalis presents a bigger and more diverse accessory genome, which probably reflects its capacity to colonize different mammalian hosts unlike C. hyointestinalis subsp. lawsonii that is host-restricted. This greater plasticity in the accessory genome of C. hyointestinalis subsp. hyointestinalis correlates to a higher incidence of genome-wide recombination events, that may be the underlying mechanism driving its diversification. Concordantly, both subspecies present distinct patterns of gene families involved in genome plasticity and DNA repair like CRISPR-associated proteins and restriction-modification systems. Together, our results provide an overview of the genetic mechanisms shaping the genomes of C. hyointestinalis subspecies, contributing to understand the biology of Campylobacter species that are increasingly found as emerging pathogens.

genomics↗