bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.07.21.214171

Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection

Kruse, R. L.·Legras, X.·Barzi, M.
View original source ↗Export metadata ↓

Abstract

New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2,753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P<0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kruse, R. L., Legras, X., Barzi, M.. 2020-07-21. Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection. https://doi.org/10.1101/2020.07.21.214171

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Microbially conjugated bile acids differentially affect the lifecycle and pathogenesis of Clostridioides difficile

Clostridioides difficile is a Gram-positive bacterium that causes C. difficile infection (CDI). In healthy adults, it has been proposed that the gut microbiota provides colonization resistance against C. difficile by competition through nutrient limitation and antagonism through secondary bile acid metabolism, the latter of which is mediated by the deconjugation of host-derived bile acids with bacterial bile salt hydrolases (BSH). Recently, BSHs have also shown an ability to reconjugate non-canonical (i.e., not glycine or taurine) amino acids to sterol cores, resulting in novel microbially conjugated bile acids (MCBAs). MCBAs have been associated with the resolution of recurrent CDI, though the mechanism underlying these effects are not well understood. To address this gap, we synthesized 36 MCBAs with nine amino acids (cysteine, isoleucine, leucine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine) conjugated to the primary and secondary bile acids found in the human gut (CA, DCA, CDCA, and LCA) and tested their impact on various stages of C. difficiles lifecycle including bacterial outgrowth, membrane integrity, sporulation, and toxin activity. We show that BSH-mediated amidation of bile acid sterol cores can either increase or decrease inhibitory effects on C. difficile in an amino acid-dependent manner. For example, serine MCBAs enhanced C. difficiles ability to grow in otherwise inhibitory bile acid concentrations, increased spore production, and elevated toxin activity. Tryptophan MCBAs increased inhibition. By demonstrating these variable effects, our work clarifies the molecular determinants of MCBA activity and highlights specific MCBAs as promising targets for the development of C. difficile microbial-based therapeutics.

microbiology↗
bioRxiv2026

Magnetic organelle gene clusters enable bacterial growth and ATP homeostasis under oxidizing conditions

Biological organisms can produce magnetic nanoparticles made of magnetite to detect the Earth's magnetic field for orientation. The evolutionary mechanisms that led to the acquisition of this capacity remain unknown. The most ancient organisms known to perform magnetic orientation are magnetotactic bacteria. Under tight genetic control, they produce chains of magnetite nanoparticles in dedicated organelles to allow efficient navigation in aquatic environments containing molecular oxygen. Yet, these bacteria have been proposed to emerge before molecular oxygen became abundant on Earth, at a time when this navigation system could not have served its current function. Here, we show that gene clusters responsible of magnetite formation and organization enable resistance against hydrogen peroxide, a toxic oxygen-derived compound that was produced during the early stages of Earth's oxygenation when iron oxidation consumed molecular oxygen. Characterization of the nanoparticle's oxidation state supported the involvement of magnetite in resistance to hydrogen peroxide-induced oxidative stress. These findings are compatible with an early emergence of magnetotactic bacteria, before natural waters became oxygenated. We propose a conceptual framework to explain the origin of magnetic orientation in bacteria, and offer tools to identify the initiation of Earth's oxygenation in a time-resolved manner.

microbiology↗
bioRxiv2026

Identification and characterization of new P. aeruginosa phages using a surface receptor platform

Bacteriophages are of increasing interest for their potential use as alternatives to antibiotics for treating drug-resistant infections. Phage steering, specifically receptor-based steering, involves use of phages that bias the development of resistance in bacteria towards phenotypes such as reduced fitness. Phage therapy typically involves administering cocktails of phages with different targets to reduce the likelihood of resistance emerging. To ensure effective steering and cocktail design, there is a need to identify the receptors used by phages of interest, and to isolate phages that use diverse receptors. Here we describe a SURFAce Receptor (SURFAR) platform consisting of a set of defined Pseudomonas aeruginosa strains and mutants for rapid characterization of phage receptors. This platform can also be used to enrich for and isolate diverse phages from environmental samples, yielding novel phage genomes from wastewater. Using the SURFAR platform, two new phages were isolated and characterized: Dalewood, which targets both flagella and the conserved LPS outer core, and Bowman, a biofilm targeting phage capable of supressing growth of a subset of carbapenem-resistant clinical isolates. This tool helps to standardize the laborious but important task of phage receptor characterization, with the goal of expanding the known repertoire of P. aeruginosa phage receptors and phages.

microbiology↗